Technology to inhibit vascular changes that lead to vision loss in the eye

ABSTRACT

This invention is based on the discovery that many eye conditions associated with aging are mediated at least in part by cells bearing a senescent phenotype. Senescent cells accumulate with age, and express factors that contribute to the pathophysiology of age related conditions. The data show that in age-matched patients, the severity of age-related conditions correlates with the abundance of senescent cells, and that clearing senescent cells can help abrogate the condition. Small molecule drugs that remove senescent cells from affected tissue in the eye are provided that have special efficacy in treating ophthalmic conditions. They not only inhibit progression of the disease, they can also reverse some of the pathophysiology—such as neovascularization and vaso-obliteration—that lead to vision loss. These senolytic agents have an appropriate dose and specificity profile to be effective in the clinical management of previously intractable ophthalmic conditions.

RELATED APPLICATIONS

This application is a continuation of international patent applicationPCT/US2018/046553, filed Aug. 13, 2018, and claims the priority benefitof U.S. patent application 62/579,793, filed Oct. 31, 2017 and Europeanpatent application 18188799.3, filed Aug. 13, 2018. The aforelisteddisclosures are hereby incorporated herein by reference in theirentirety for all purposes.

BACKGROUND

The prevalence of adult vision impairment and blindness due toage-related eye disease is one of the largest challenges facing modernmedicine.

According to the World Health Organization, three eye conditions haveemerged as potential threats to the status of sight of people inmiddle-income and industrialized countries throughout the world. Theincrease in the prevalence of Type II diabetes has caused diabeticretinopathy to take top place on the WHO's priority list. Glaucoma, adisabling eye disease known for centuries, remains on the public healthagenda due to difficulties in its early diagnosis and frequent necessityof life-long treatment. Age-related macular degeneration (AMD) ranksthird among the global causes of visual impairment with a blindnessprevalence of 8.7%. It is the primary cause of visual impairment inindustrialized countries.

According to the National Eye Institute (NEI), in the U.S. population2.1 million have age-related macular degeneration, 7.7 million havediabetic retinopathy, 2.7 million have glaucoma, and 24.4 million havecataracts. This represents a remarkable 26% of the American populationover 40 years of age.

In spite of the prevalence of visual disability, and all the attentionit receives in the medical research community, these disorders remainlargely intractable. Many conditions have no available ordisease-modifying therapeutic alternatives. With few exceptions, mostdrugs currently approved for treating these disorders are directed atlate-stage pathophysiology or the relief of symptoms, rather thanaddressing the factors that initiate and/or maintain the disease.

Currently available modes of therapy include inhibitors of vascularendothelial growth factor (VEGF) agents for the treatment ofVEGF-associated eye disease (for example, wet AMD, diabetic eyedisease), vitamins and anti-oxidants for dry AMD, and agents that lowerintraocular pressure for glaucoma. Laser treatments are available totreat some conditions: for example, retinal photocoagulation for retinaledema or neovascularization secondary to diabetes, vein occlusion, andchoroidal neovascularization; and laser trabeculoplasty to addresselevated intra-ocular pressure resistant to medical therapy. For manyocular disorders including dry AMD, there are no currently approvedtherapeutic agents. A Phase 3 clinical trial for a humanized antibodydesigned for treatment of geographic atrophy in dry AMD (lampalizumab)recently failed to meet its primary endpoint of preventing atrophyprogression. Even for eye diseases where therapeutic agents areavailable, the treatment regimens are often burdensome and have limitedlong term efficacy.

The invention provided here creates a new paradigm for the treatment ofeye disease through the elimination of senescent cells implicated in thepathophysiology of disorders of the visual system. The disclosure thatfollows outlines its implementation and use, and describes many of theensuing benefits.

SUMMARY

This invention is based on the discovery that many eye conditionsassociated with aging are mediated at least in part by cells bearing asenescent phenotype. Senescent cells accumulate with age, and expressfactors that contribute to the pathophysiology of age relatedconditions. The data show that in age-matched patients, the severity ofage-related conditions correlates with the abundance of senescent cells,and that clearing senescent cells can help abrogate the condition.

Small molecule drugs that remove senescent cells from affected tissue inthe eye are provided as part of this invention that have specialefficacy in treating ophthalmic conditions. They not only inhibitprogression of the disease, they can also reverse some of thepathophysiology—such as neovascularization and vaso-obliteration—thatlead to vision loss. These senolytic agents have an appropriate dose andspecificity profile to be effective in the clinical management ofpreviously intractable ophthalmic conditions.

In general terms, this invention provides technologies for preventing ortreating an ophthalmic condition in a subject by removing senescentcells in or around an eye of the subject so that progression of thecondition is delayed or at least one sign or symptom of the disease isdecreased in severity.

For purposes of this disclosure, ophthalmic diseases are classifiedaccording to six general types of pathophysiology: an ischemic orvascular condition; a degenerative condition; a genetic condition; abacterial, fungal, or virus infection; an inflammatory condition; or aniatrogenic condition. The underlying pathophysiology is instructive inthe implementation of a senolytic strategy to treat each disease.Classification of ophthalmic diseases within these types is provided inthe disclosure that follows.

Included in the invention are methods of treatment, unit doses, anddedicated uses of particular inhibitors and senolytic agents. Effectiveagents that can be used in the context of this invention to removesenescent cells and treat ophthalmic conditions include compounds havingthe following formula, or a phosphorylated form thereof:

wherein:

-   -   R₁ and R₂ are independently C₁ to C₄ alkyl    -   R₃, R₄ and R₅ are independently —H or —CH₃;    -   R₈ is —OH or —N(R₆)(R₇), wherein R₆ and R₇ are independently        alkyl or heteroalkyl, and are optionally cyclized;    -   X₁ is —F, —Cl, —Br, or —OCH₃;    -   X₂ is —SO₂R′ or —CO₂R′, where R′ is —H, —CH₃, or —CH₂CH₃;    -   X₃ is —SO₂CF₃; —SO₂CH₃; or —NO₂; and    -   X₅ is —F, —Br, —Cl, —H, or —OCH₃.

In some implementations, X₃ is —SO₂CF₃ or —NO₂, and R₈ is —N(R₆)(R₇),wherein R₆ and R₇ are independently alkyl or heteroalkyl, and areoptionally cyclized.

Depending on how the technology is implemented, besides generallyimproving symptomatology or preventing advancement of a particularophthalmic condition, the technology may have one or more of thefollowing effects in any combination:

-   -   reducing the number of p16 positive senescent cells in or around        an eye of the subject;    -   inhibiting or reversing neovascularization in an eye of the        subject;    -   inhibiting or reversing vaso-obliteration in an eye of the        subject; and    -   inhibiting or reversing increased intra-ocular pressure (IOP) in        an eye of the subject.

Also provided as part of this invention are novel screening methods. Onesuch method comprises selecting a test agent as a possiblepharmaceutical compound for treating glaucoma by contacting trabecularmeshwork (TM) cells in culture or in a tissue of a non-human testsubject, and determining whether the test agent reduces the number ofsenescent cells in the culture or tissue. Another such screening methodcomprises administering a test agent to an eye of a non-human testsubject, and determining whether the test agent inhibits or reversesneovascularization or vaso-obliteration caused in the course of ananimal disease model.

Other features of the technology of the invention are provided in thesections below and in the appended claims.

DRAWINGS

FIG. 1A is a flow chart that shows the pathophysiologic interactions inthe eye that result from ischemia. FIG. 1B is a flow chart that showsthe multifactorial pathophysiology of glaucoma, leading to retinalganglion cell (RGC) cell death. FIG. 1C is a flow chart that shows thepathophysiologic cascade in age related macular degeneration. FIG. 1D isa flow chart that shows events leading to cell degeneration and celldeath in Leber's Hereditary Optic Neuropathy.

FIG. 2A comprises images of histopathology that show retinal cell lossin stages of Retinitis Pigmentosa. FIGS. 2B, 2C, 2D, and 2E arefluorescein angiograms of a normal retina and retinal non-perfusion andneovascularization from diabetes, sickle cell disease and inflammatoryvasculitis, respectively.

FIGS. 3A and 3B show nine particular compounds selected from a libraryon the basis of binding to Bcl-2 or Bcl-xL.

FIGS. 4A, 4B, and 4C show quantitative binding affinity of the ninecompounds to Bcl isoforms Bcl-xL, Bcl-2, and Bcl-w, respectively. Eachof the compounds for which the data is shown are identified according totheir designated BM number.

FIGS. 5A, 5B, and 5C shows how effective compounds were comparedstructurally to determine what substructures contribute to the desiredproperties of the compounds.

FIG. 6 shows binding affinity to Bcl isoforms, and the effectiveconcentration (EC₅₀) for killing senescent fibroblasts (SnCs) inculture.

FIG. 7 is a concentration-response curve for senescent human retinalmicrovascular endothelial cells HRMEC cells and control cells treated invivo with a senolytic agent.

FIG. 8 is a concentration-response curve for senescent retinal pigmentepithelium (RPE) cells and control cells treated in vivo with asenolytic agent. The agent has a much higher potency (lower LD₅₀) forthe senescent cells than for normal proliferating RPE cells. It has aselectivity index for senescent RPE cells compared with non-senescentRPE cells of between 10 and 100.

FIGS. 9A and 9B show reversal of both neovascularization andvaso-obliteration in the mouse oxygen-induced retinopathy (OIR) modelwhen intravitreally administered with the senolytic agent UBX1967.

FIGS. 10A, 10B, 10C, and 10D show decreased expression levels at the RNAtranscript level of senescence-associated markers, following treatmentwith UBX1967.

FIGS. 11A and 11B are taken from the streptozotocin (STZ) model fordiabetic retinopathy. STZ-induced vascular leakage is attenuated withthe intravitreal administration of UBX1967.

FIGS. 12A and 12B show immunohistochemistry staining for p16 (a markerfor senescent cells) in tissue taken from a patient with primary openangle glaucoma (POAG). p16 positive cells are prominent in thetrabecular meshwork (TM).

FIGS. 13A and 13B show the expression of p16 in human eye tissueobtained from donors diagnosed with primary open angle glaucoma (POAG).

FIGS. 14A and 14B show immunohistochemistry staining for p16 in humanretinal tissue of a patient with age-related macular degeneration (AMD).

DETAILED DESCRIPTION

Overview

It is a premise of this disclosure that many or most ophthalmicconditions that are age-related, or are associated with cellular defectsthat lead to an accelerated aging phenotype, are caused or mediated atleast in part by senescent cells, which accumulate with age and withdeleterious impact on ophthalmic tissues. Senescent cells are typicallycells that no longer have replicative capacity, but remain in the tissueof origin, eliciting a senescence-associated secretory phenotype (SASP).Senescent cells are thought to derive from proliferative cells of avariety of tissue types, including cells that reside in and around theeye. SASP factors include molecules that are angiogenic, inflammatory,fibrotic, and extracellular matrix modifying molecules (Acosta et al.,2013). Some factors implicated in ocular pathologies are part of theconstellation of factors produced by senescent cells. For this reason,elimination or control of senescent cells provides a means by which totreat eye disease, not only through the elimination of senescent cellsbut also through reduction of their associated SASP factors and impacton surrounding cells.

Different eye conditions present in the clinic with different signs andsymptoms, and have different types of pathophysiologic mechanisms. Theheterogeneity of eye conditions is consistent with the putative role ofsenescent cells in the disease pathology, because senescent cells may befrom different cell lineages, induced by different stressors, reside indifferent ocular tissues, and interact with surrounding cells in adifferent fashion. Nevertheless, senescent cells in the various tissuesof the eye have a related secretory phenotype that contributes todisorders throughout the visual system. The specific clearance ofsenescent cells from tissue is referred to in this disclosure assenolysis. Small molecule compounds capable of senolysis are referred toas senolytic agents, and clear senescent cells irrespective of mechanismof senescence induction, SASP profile or cell lineage.

By way of illustration, we have found that inhibitors of the Bcl familyof proteins trigger apoptosis in senescent cells derived from a celltype known to reside in the back of the eye and are cells implicated inretinal disease such as AMD—and also trigger apoptosis in senescentcells derived from a cell type known to reside in the anteriorcompartment of the eye and are cells implicated in diseases such asglaucoma.

Discoveries that Change the Current Paradigm for Drug Development

Besides a new understanding of the general role of senescent cells inmediating ophthalmic conditions, other discoveries are described in thisdisclosure that open new avenues for drug development.

One is the discovery that senescent cells are abundant in the trabecularmeshwork of patients with glaucoma, and in corresponding animal models.Intraocular pressure increase is caused by either an overproduction ofaqueous humor primarily in the ciliary body, or a decrease in outflow ofaqueous fluid primarily through the trabecular meshwork The datapresented here are consistent with the idea that at least part of thepathophysiology underlying the disease is impaired drainage ofintraocular fluid through the trabecular meshwork and down the canal ofSchlemm and the episcleral veins into the orbital venous system.Targeting senolytic drugs to cells in the trabecular network is animportant new avenue for pharmaceutical development: both asmonotherapies, and in combination with drugs that work by regulatingfluid production.

Another discovery is that removing senescent cells from the back of theeye in diseases such as diabetic retinopathy doesn't just inhibitdisease progression: it actually reverses some of the pathophysiologythat leads to loss of vision, including neovascularization andvaso-obliteration. To our knowledge, there is no current therapy (eitherin the clinic or in development) that is able to reverse the course ofretinopathy to this extent. The objective of therapy for ophthalmicconditions can now be more ambitious—giving renewed hope to patientswith these diseases for an improved quality of life.

Advantages of Treating Ophthalmic Conditions by Clearing Senescent Cells

The role of senescent cells in promoting or mediating a variety ofophthalmic conditions provides an approach to treatment with a number ofadvantages for the managing clinician.

-   -   Since senescent cells are non-proliferative, eliminating        senescent cells has the potential for a clinically beneficial        effect that persists for an extended time between episodes of        treatment. Features of the condition mediated by senescent cells        resolve at least until senescent cells re-accumulate. Since        senescent cells are likely to accumulate slowly (given the        nature of age related diseases is to evolve over a period of        many years), the effects of a single treatment or treatment        cycle may last for weeks, months, or years.    -   To the extent that senescent cells exacerbate other types of        pathology such as inflammation or tissue breakdown, the        long-lasting effect of senolysis provides a window in which such        pathology is held at bay, potentially giving the tissue a chance        for repair. This means that senescent cell medicine has the        potential not just to halt progression of ophthalmic conditions,        but allow some degree of reversal of the disease and its        symptoms for the benefit of the patient.    -   Since senescent cells in different parts of the eye respond to        the same senolytic agents, several different eye diseases can be        treated in the same patient at the same time. For example, a        patient may present to the clinician with several disease        processes already under way: such as glaucoma and macular        degeneration. It may be possible to administer a single        senolytic agent in a treatment protocol that addresses the        disease and its symptoms of each of the multiple conditions.        Beyond the convenience of this approach, it has the added        benefit of lowering the risk of side effects that may result        from multiple drugs being given in combination to treat each of        the conditions individually. Furthermore, it is possible that        factors elicited by cells in one part of the eye may impact        other parts of the eye such that treating senescence to two        locations may have a beneficial effect on both diseases.    -   By addressing the early pathology in a disease, senolytic        medicine can be an important adjunct to other types of therapy        that are administered to treat later stage pathology, or to        relieve the symptoms that result from the condition. The two        modes of therapy potentially work synergistically or additively        to reduce the burden, frequency and side effects of either mode        administered separately.        Classification of Eye Disease According to Underlying        Pathophysiology

As a guide to treating ophthalmic conditions in accordance with thisinvention, the conditions can be classified according to the primaryunderlying pathophysiology. Conditions that fall within the sameclassification are amenable to applying senolytic medicine with the sameprinciples and with similar objectives.

Ophthalmic conditions suitable for treatment are discussed in moredetail below, within the following classifications:

-   -   TYPE 1: Ischemic or vascular conditions: result from a        restriction in blood supply to tissues, causing a deficiency of        oxygen and/or essential nutrients needed for cellular metabolism        to keep tissue functional.    -   TYPE 2: Degenerative conditions: characterized by a progressive        deterioration in quality, function, or structure of a tissue or        organ, leading to progressive visual impairment.    -   TYPE 3: Genetic conditions: caused by a mutation, deletion, or        insertion in an individual's DNA sequence.    -   TYPE 4: Infectious conditions, caused by pathogenic        microorganisms, such as bacteria, viruses, parasites or fungi;        the diseases can be spread, directly or indirectly, from one        person to another.    -   TYPE 5: Inflammatory conditions characterized by a localized        response elicited by injury, foreign object, or destruction of        tissues, which serves to destroy, dilute, or wall off both the        injurious agent and the injured tissue via the production of        pro-inflammatory mediators and recruitment of immune system        cells.    -   TYPE 6: Iatrogenic conditions, defined as disease that is the        result of diagnostic and therapeutic procedures undertaken on a        patient.

This classification is provided to assist the reader in understandingand applying the invention to a particular patient, and is not meant tolimit application of this technology. Certain conditions may invokeseveral of these categories: for example, an inflammatory process maycontribute to pathological processes having other underlying causes.Similarly, the SASP may trigger additional pathologic processesregardless of the primary insult.

Treatment Design

Senescent cells accumulate with age, which is why conditions mediated bysenescent cells occur more frequently in older adults. In addition,different types of stress on ocular tissues may promote the emergence ofsenescent cells and the phenotype they express. Cell stressors includeoxidative stress, metabolic stress, DNA damage (for example, as a resultof environmental ultraviolet light exposure or genetic disorder),oncogene activation, and telomere shortening (resulting, for example,from hyperproliferation). Ocular tissue subject to such stressors mayhave a higher prevalence of senescent cells, which in turn may lead topresentation of certain eye diseases at an earlier age, or in a moresevere form. An inheritable susceptibility to certain eye diseasessuggests that the accumulation of disease-mediating senescent cells maydirectly or indirectly be influenced by genetic components, which canlead to earlier presentation.

To treat a particular ophthalmic condition with a senolytic agentaccording to this invention, the therapeutic regimen will depend on thelocation of the senescent cells, and the pathophysiology of the disease.

With respect to location, disorders of the visual system are broadlyclassified as anterior and posterior. An anterior ocular condition is adisease, ailment or condition that affects or involves an anteriorocular region or site, such as periocular muscle, eyelid or eye tissueor fluid which is located anterior to the posterior wall of the lenscapsule or ciliary muscles. A posterior ocular condition is a disease,ailment or condition which primarily affects or involves a posteriorocular region or site such as chorioid, ciliary body, vitreous, vitreouschamber, retina, retinal pigment epithelium, Bruch's membrane, opticnerve (i.e. the optic disc), visual pathway and blood vessels and nerveswhich vascularize or innervate a posterior ocular region or site.

With respect to pathophysiology, senescent cells and SASP production cancontribute to ongoing cell dysfunction and degeneration/death. Senescentcells and their associated SASP factors can mediate the associatedcontributions to ongoing cell dysfunction, cell loss, and diseaseprogression via blockage of the angiogenic, inflammatory, fibrotic, andextracellular matrix-modifying proteins present in the pathophysiology.

Thus, elimination or reduction in the number of senescent cells in oraround the site of the pathology removes at least one of the causes ormediators of the condition, as it is manifested in the clinic. Thesenolytic agent is formulated and administered in such a way that itcontacts senescent cells in or around the site of the pathology,clearing them and/or inhibiting their activity to an extent that haltsprogression of the condition and/or signs and symptoms of the conditionare relieved.

Different conditions mediated by senescent cells in about the samelocation and/or with a similar underlying pathology will often betreated in the same way. For example, a senolytic agent can beadministered by local topical administration to the site affected indisorders of the anterior segment e.g., to the conjunctiva and/orcornea, for example using eye drops, ointment, or via application of acontact lens. Intraocular injection, either intracameral or intravitrealcan be administered for disorders of both the anterior and posteriorsegments.

Diagnosis and Monitoring of Disorders of the Visual System:

The approach to the diagnosis and monitoring of all ocular conditionsand diseases, and evaluation of therapeutic effects, is facilitated bythe widespread availability of a standard battery of tests of ocularstructure and function. These tests can evaluate individual layers ofthe eye extending from the lids and anterior segment to the vitreous andall retinal cell layers, the optic nerve and the visual cortex.

Standardized ophthalmic examination includes a detailed slit lampbiomicroscopic evaluation which allows evaluation of the lids, ocularadnexa, lashes, corneal surface, anterior chamber, pupils, lens,vitreous cavity and central retinal anatomy including the optic nerveand macula. Gonioscopy allows detailed examination of the anteriorchamber angle, important in the diagnosis and monitoring of all forms ofglaucoma. Indirect ophthalmoscopy allows for evaluation of the retinalperiphery, important in the monitoring of vitreous and peripheralretinal disorders.

Ancillary testing is also widely used in the diagnosis and monitoring oftherapeutic response in all disorders of the visual system and thesetests include the following:

Functional tests of visual acuity (including best corrected acuity,contrast acuity, and low luminance acuity), color vision (includingIshihara and Farnsworth Munsell tests) and visual field evaluation(including Humphrey automated perimetry and microperimetry), tearproduction (Schirmer test), and tonometry to measure intraocularpressure (IOP). These are used in conjunction with structural testsincluding anterior and posterior segment photographs, cornealpachymetry, ultrasound, ultrasound biomicroscopy, optical coherencetomography (OCT), intravenous fluorescein angiography (IVFA), and fundusautofluorescence (FAF). Imaging such as computerized tomography (CT) ormagnetic resonance imaging (MRI) scans are utilized to evaluate ocular,periocular and orbital structures, and the intracranial portion of theoptic nerve, visual pathway and visual cortex in the brain. These testsallow visualization of structural integrity and thickness of the layersof the eye and surrounding structures, and assessment of blood flow andcirculation.

Advanced functional testing of the retina, optic nerve and visualpathway/cortex is also used, including electrophysiologic tests such asfull field and multifocal electroretinography, visual evoked potentialsand microperimetry to diagnose and monitor disease progression andimpact of therapy (Mengini and Duncan. 2014). While currently availableonly in research settings, other imaging technologies may becomeimportant adjuncts in the diagnosis and treatment of disease of the eye,including those due to senescence, such as the use of adaptive opticsand optical coherence angiography.

Clinical examination, structural and functional measurements andcorrelations can be obtained in both animal models and the clinicalsetting and are applicable to the conditions and diseases of the visualsystem outlined in this application. The battery of tests as outlinedabove is part of the diagnosis, evaluation and response to treatment forthese conditions.

As examples, retinal non-perfusion and neovascularization seen secondaryto retinal ischemia produced by a range of different etiologic factors(e.g. diabetic retinopathy, vascular occlusive disease due toatherosclerotic or inflammatory causes, retinopathy of prematurity andgenetic vascular disorders such as Sickle Cell retinopathy) can bestructurally evaluated by IVFA and OCT to both diagnose and monitorresponse to a senolytic. Glaucomatous optic neuropathy with loss ofretinal ganglion cells and visual field function that are the result ofoptic nerve susceptibility to increased IOP stemming from a host ofcauses (for example, remodeling of trabecular meshwork, primary openangle glaucoma (POAG), pseudoexfoliation, pigmentary dispersion, steroidtreatment, trauma) can be diagnosed and monitored by OCT and visualfield testing. The role of senescent cells as identified by molecularmarkers such as p16 in trabecular meshwork tissue of glaucoma patients(Example 5) and in retinal tissue in donor eyes with AMD (Example 6),and the presence of SASP factors known to be implicated in variousstages of these diseases, highlight the potential impact of senolyticmedicine on these conditions.

A consequence of this invention is that regardless of the exact means bywhich senescent cells accumulate and subsequently express SASP,senolytic therapy can have a beneficial impact on ocular diseasefeatures through the restoration of homeostasis in the cellular milieu.This results in disease modification via a change in the disease courseand outcome.

Comparison of Senolytic Medicine with Currently Available Therapy

Therapies that are currently in clinical use are limited in theirability to achieve disease modification or potential reversal ofpathology. The standard of care for the most prevalent ocular diseases(glaucoma and retinal and choroidal vascular disease) are topical dropsto lower intraocular pressure (IOP) in glaucoma, intra-ocular injectionof anti-VEGF agents for retinal and choroidal neovascular disease, andlaser photocoagulation for both IOP control (Stein and Challa, 2007) andvitreo-retinal disorders (AAO Retina/Vitreous Panel, 2014).

Topical agents that lower intraocular pressure (IOP) and therapiestargeting VEGF-related eye diseases, are burdened by a frequentadministration schedule that must be adhered to in order to maximizeefficacy. Even when administered optimally, anti-VEGF therapies areassociated with a significant rate of incomplete response, diseaserecurrence, and ongoing progression of non-VEGF mediated aspects of theocular disease (for example atrophy of the macula in treated wet AMD(Bhisitkhul, 2015). These same issues are a concern for IOP loweringagents used in glaucoma, which must be administered at least daily tolower IOP, and have been associated with ongoing glaucomatous diseaseprogression even when used appropriately and associated with decreasedIOP (Levin, 2005).

Laser photocoagulation has been another mainstay of ocular therapy usedacross a wide range of ocular diseases, over which senolyticadministration can have many advantages. Although clinically effective,retinal laser photocoagulation leads to collateral damage and sideeffects including reduced night vision, macular and peripheral scotomatawith decrease in central and peripheral vision, exacerbation of macularedema and disruption of the retinal anatomy through scarring (Kozak andLuttrul, 2015). In its application to the trabecular meshwork to lowerIOP, laser therapy is associated with IOP spikes, peripheral anteriorsynechiae formation, need for additional laser or surgical procedures,and no reduction in the need for IOP-lowering drops following theprocedure (Damji et al., 2006).

Removal of senescent cells and the associated SASP with senolytictherapy can positively impact disease course via the modulation ofmultiple disease-mediating factors including inflammatory, angiogenicand extracellular matrix-modifying aspects of the disease. With limitedor no damage or destruction to healthy cells required to maintain visualfunction, and an infrequent dosing schedule with prolonged therapeuticeffect this invention represents a major advance over currentlyavailable therapies that do not specifically target senescent cells orthe multiple factors associated with the SASP, thus limiting theirability to modify multiple aspects of disease pathophysiology.

As an example, the elimination of senescent cells in the setting ofischemia can impact visual function by allowing functioning retinalganglion cells to thrive in a healthier local environment, free of theSASP associated detrimental inflammatory, angiogenic and extracellularmatrix-modifying factors. This can be monitored structurally by opticalcoherence tomography (OCT) measurements of retinal thickness, andfunctionally by electrophysiology testing (VEP and ERG) that can isolatefunction of the retinal ganglion cell layer. Automated perimetry canalso be used to evaluate peripheral visual field function in patients.

Similarly, in the setting of vaso-obliterative disease andneovascularization, the removal of senescent cells and SASP canpotentially ameliorate ongoing associated cell damage and allow forre-perfusion of the affected vascular beds and decreased neovasculardrive. The similarity of the ischemic phenotype (vaso-obliteration andneovascularization) regardless of etiology can be evaluated, andstructural and functional response to therapy monitored by IVFA, OCT,and ERG. FIG. 2B (Image from Carver College of Medicine, University ofIowa website, sourced Oct. 30, 2017), FIG. 2C (Image from Retina Gallerywebsite, sourced Oct. 30, 2017), FIG. 2D (Image from Retina VitreousAssociates of Florida website, sourced Oct. 30, 2017), and FIG. 2E(Image from Retina Gallery website, sourced Oct. 30, 2017) demonstratefluorescein angiographic examples of a normal retina (FIG. 2B) andretinal non-perfusion and neovascularization from diabetes (FIG. 2C),sickle cell disease (FIG. 2D) and inflammatory vasculitis (FIG. 2E).Response to senolytic therapy can be monitored with intravenousfluorescein angiography (IVFA) and OCT. Factors known to be involved inthe SASP of senescent cells may be measured directly in ocular fluids,including tears, aqueous humor and vitreous humor.

The impact of senolytic medicine on the treatment of eye diseaseincorporates three main concepts. First, once senescent cells aredeleted, it is expected that the associated SASP factors derived fromsenescent cells will also be greatly diminished. In the absence of theseinflammatory, angiogenic, and fibrotic proteins and extra-cellularmatrix modifying enzymes, it is postulated that many or most of thesymptoms of the ocular diseases described herein can be greatlyimpacted. Importantly, after senescent cells are deleted, it is possiblethat surrounding cells can restore some functional capacity. Thisrepresents a major pathophysiologic advance over currently availabletreatments for ocular disease. The impact of the senolytic agent andrestoration of function can be monitored clinically with structural andfunctional testing such as outlined above.

Second, depending on the circumstances, a senolytic agent can bedelivered as a single administration. If retreatments are necessary, thetime between doses is considerably extended. As macular degeneration,glaucoma, and vascular and hereditary retinopathies are characterized byslow degradation of the retina over a period of many years,re-accumulation of senescent cells takes a substantial period of time.Further therapy may not be needed for several years. This represents amajor improvement over for example the dosing schedule of existinganti-VEGF therapies which require once monthly to every other monthadministration and are associated with suboptimal visual and anatomicoutcomes if delivered on a less frequent schedule (Maguire et al. 2016,Holz et al. 2014). Topical IOP lowering agents require dailyadministration and a significant percentage of patients demonstrateglaucomatous progression despite IOP lowering (Levin, 2005).

Finally, senolytic therapy of eye diseases may address an underlyingcommon mechanistic cause of the ocular disease rather than impactingonly symptoms generated from downstream signaling pathways. A senolyticagent can for example impact on multiple pathology-associated cytokines(inflammatory, cytotoxic, angiogenic, fibrotic) rather than the specificinhibition of a single factor involved in a single aspect of the disease(for example, anti-VEGF therapy for the VEGF related aspect ofneovascularization).

As an example, senolytic therapy can reduce a range of growth factorsknown to be implicated in various stages of AMD. FIG. 1C (Kumar and Fu,2014) indicates that early deposits in the RPE impact the degenerationof the extracellular matrix including elastin and fibronectin.Increasing oxidative stress induces mitochondrial DNA damage, a knownpotent inducer of senescence. Additional activation of pro-inflammatorycytokines and chemokines (IL-1, IL-6) follows, with eventual inductionof VEGF and metalloprotease (MMPs) and inflammosome activation. Asenolytic therapy that targets this range of factors (all identified ascomponents of the SASP) can exert a multi-pronged impact along thepathophysiologic course of AMD, with the ability to modulate andpotentially reverse the course of disease. True disease modification hasnot to date been demonstrated by available therapies for oculardisorders, highlighted by the necessity for frequent administration tocontrol the diseases. The potential for infrequent dosing and diseasemodification offered by senolytic therapy represents a major advance inocular disease therapy.

Senescent cell deletion may be a disease modifying treatment for oculardiseases arising from ischemia, degeneration or genetic root causes, byeither halting progression or potentially allowing endogenous reparativesystems or improved cell function to modify outcomes.

Suitable Senolytic Agents

Compounds that may be useful for clearing senescent cells in or near theeye for purposes of treating ophthalmic conditions according to thisinvention include Bcl-2 inhibitors, Bcl-xL inhibitors, MDM2 inhibitors,and Akt inhibitors. See U.S. Pat. Nos. 8,691,184, 9,096,625, and9,403,856; published applications WO 2015/17159, WO 2015/116740, WO2016/127135, and WO 2017/008060; and unpublished applicationPCT/CN2016/110309.

Candidate senolytic agents that act as Bcl-2, Bcl-w, and Bcl-xLinhibitors can be characterized as a benzothiazole-hydrazone, an aminopyridine, a benzimidazole, a tetrahydroquinolin, or a phenoxyl compound.Examples of compounds that inhibit Bcl isoforms include WEHI 539, A1155463, ABT 737, and ABT 263 (Navitoclax).

Candidate senolytic agents that act as MDM2 inhibitors can becharacterized as a cis-imidazoline, a dihydroimidazothiazole, aspiro-oxindole, a benzodiazepine, or a piperidinone. Candidate in MDM2include Nutlin-1, Nutlin-2, Nutlin-3a, RG-7112, RG7388, R05503781,DS-3032b, MI-63, MI-126, MI-122, MI-142, MI-147, MI-18, MI-219, MI-220,MI-221, MI-773,3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one,Serdemetan, AM-8553, CGM097, R0-2443, and R0-5963.

Candidate senolytic agents that act as inhibitors of Akt (protein kinaseB) are the competitive Akt inhibitors CCT128930, GDC-0068, GSK2110183(afuresertib), GSK690693, and AT7867; the lipid-based Akt inhibitorsCalbiochem Akt Inhibitors I, II and III, PX-866, and Perifosine(KRX-0401); the pseudosubstrate inhibitors vKTide-2 T and FOXO3 hybrid;allosteric inhibitors of the Akt kinase domain, particularly MK-2206(8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-2H-[1,2,4]triazolo[3,4-f][1,6]naphthyridin-3-one;dihydrochloride); the antibody GST-anti-Akt1-MTS; the compounds thatinteract with the PH domain of Akt Triciribine and PX-316; and othercompounds exemplified by GSK-2141795, VQD-002, miltefosine, AZD5363,GDC-0068, and API-1.

Exemplary Bcl inhibitors for use in treating ophthalmic conditionsaccording to this invention contain a structure according to Formula Ias shown below, or a phosphorylated form thereof.

wherein:

-   -   R₁ and R₂ are independently C₁ to C₄ alkyl    -   R₃, R₄ and R₅ are independently —H or —CH₃;    -   R₈ is —OH or —N(R₆)(R₇), wherein R₆ and R₇ are independently        alkyl or heteroalkyl, and are optionally cyclized;    -   X₁ is —F, —Cl, —Br, or —OCH₃;    -   X₂ is —SO₂R′ or —CO₂R′, where R′ is —H, —CH₃, or —CH₂CH₃;    -   X₃ is —SO₂CF₃; —SO₂CH₃; or —NO₂    -   X₅ is —F, —Br, —Cl, —H, or —OCH₃.

Optionally, R₈ is —N(R₆)(R₇), wherein R₆ and R₇ are independently alkylor heteroalkyl, and are optionally cyclized.

Optionally, R₁ and R₂ are independently C₁ to C₄ alkyl;

-   -   R₃ and R₄ are both —H;    -   R₅ is —H or —CH₃;    -   R₆ and R₇ are independently alkyl or heteroalkyl, and are        optionally cyclized;    -   X₁ is —F or —Cl;    -   X₂ is —SO₂R′ or —CO₂R′, where R′ is —H, —CH₃, or —CH₂CH₃;    -   X₃ is —SO₂CF₃ or —NO₂; and    -   X₅ is —F or —H;

Other exemplary Bcl inhibitors for use in treating ophthalmic conditionsaccording to this invention contain a structure according to Formula II,as shown below, or a phosphorylated form thereof.

wherein:

-   -   R₁ and R₂ are independently C₁ to C₄ alkyl;    -   R₃ and R₄ are independently —H or —CH₃;    -   R₈ is —OH or

-   -   X₁ is —F, —Cl, —Br, or —OCH₃;    -   X₂ is —SO₂R′ or —CO₂R′, where R′ is —H, —CH₃, or —CH₂CH₃;    -   X₃ is —SO₂CF₃; —SO₂CH₃; or —NO₂    -   X₄ is —OH, —COOH or —CH₂OH;    -   X₅ is —F, —Cl, or —H; and    -   n₁ and n₂ are independently 1, 2, or 3.

Optionally, X₃ is —SO₂CF₃ or —NO₂, and R₈ is

wherein X₄ is —OH or —COOH.

Optionally, the compound may have one, two, three, more than three, orall of the following features in any combination:

-   -   R₁ is isopropyl;    -   R₂ is methyl;    -   R₃ is —H;    -   R₄ is —H;    -   X₁ is —Cl;    -   X₂ is —SO₂CH₃;    -   X₃ is —SO₂CF₃;    -   X₄ is —OH;    -   n₁ is 2; and    -   n₂ is 2.

Other exemplary Bcl inhibitors for use in treating ophthalmic conditionsaccording to this invention contain a structure according to FormulaIII, as shown below, or a phosphorylated form thereof.

wherein:

-   -   R₁ and R₂ are independently C₁ to C₄ alkyl;    -   R₃, R₄ and R₅ are independently —H or —CH₃;    -   R₆ and R₇ are independently alkyl or heteroalkyl, and are        optionally cyclized;    -   X₁ is —F, —Cl, —Br, or —OCH₃;    -   X₂ is —SO₂R′ or —CO₂R′, where R′ is —H, —CH₃, or —CH₂CH₃;    -   X₃ is —SO₂CF₃ or —NO₂; and    -   X₅ is —F, —Br, —Cl, —H, or —OCH₃;

or alternatively, wherein:

-   -   R₁ and R₂ are independently C₁ to C₄ alkyl;    -   R₃ and R₄ are both —H;    -   R₅ is —H or —CH₃;    -   R₆ and R₇ are independently alkyl or heteroalkyl, and are        optionally cyclized in the manner shown in Formula VII;    -   X₁ is —F or —Cl;    -   X₂ is —SO₂R′ or —CO₂R′, where R′ is —H, —CH₃, or —CH₂CH₃;    -   X₃ is —SO₂CF₃ or —NO₂; and    -   X₅ is —F or —H.

Other exemplary Bcl inhibitors for use in treating ophthalmic conditionsaccording to this invention contain a structure according to Formula IV,as shown below, or a phosphorylated form thereof.

wherein:

-   -   R₁ and R₂ are independently C₁ to C₄ alkyl;    -   R₃ and R₄ are independently —H or —CH₃;    -   X₁ is —F, —Cl, —Br, or —OCH₃;    -   X₂ is —SO₂R′ or —CO₂R′, where R′ is —H, —CH₃, or —CH₂CH₃;    -   X₃ is —SO₂CF₃ or —NO₂;    -   X₄ is —OH or —COOH;    -   X₅ is —F —Cl or —H; and    -   n₁ and n₂ are independently 1, 2, or 3.        Screening Compounds for Senolytic Activity

These and other compounds can be screened on the molecular level fortheir ability to perform in a way that indicate that they are candidateagents for use according to this invention.

For example, where the therapy includes triggering apoptosis ofsenescent cells by way of Bcl-2, Bcl-xL, or Bcl-w, compounds can betested for their ability to inhibit binding between Bcl-2, Bcl-xL, orBcl-w and their respective cognate ligand. Example 1 provides anillustration of a homogeneous assay (an assay that does not require aseparation step) for purposes of determining binding to the Bclisoforms. Compounds can be screened on the molecular level for theirability to act as agonists of MDM2, thereby promoting p53 activity andcausing senolysis. Example 2 provides an illustration of an assay forthis purpose.

Cell Culture Systems for Testing Senolytic Agents

Compounds can be screened for biological activity in an assay usingsenescent cells. Cultured cells are contacted with the compound, and thedegree of cytotoxicity or inhibition of the cells is determined. Theability of the compound to kill or inhibit senescent cells can becompared with the effect of the compound on normal cells that are freelydividing at low density, and normal cells that are in a quiescent stateat high density.

Example 3 provides an illustration using the human lung fibroblast IMR90cell line. Because of the facility of expanding IMR90 cells, they areeffective as an early screening tool. Since this disclosure reveals celltypes in the eye that generate senescent cells implicated in eyedisease, compounds selected in early screening can be rescreened usingprimary cultures of target eye cells: particularly trabecular meshworkcells, as illustrated in Example 4, and RPE cells, as illustrated inExample 5.

Where technically feasible, tissue explants from human patient donoreyes can be generated and the reduction of senescent cell and diseaserelevant markers measured after incubation with test compounds. In thisformat, senolysis, and its downstream impact, can be measured in anintact tissue, where relevant cell types are present and senescence wasdriven by disease pathogenesis. Tissue explants can also provide a meansto assess disease-relevant cell types that are not easily amenable tostandard in vitro cell culture methods in isolation (for example,photoreceptors and neurons).

Animal Models for Testing Senolytic Agents

Test compounds can be assessed in preclinical animal models to gainconfidence in target engagement of relevant cell types and downstreamreadouts such as reduction of SASP or efficacy against functionalendpoints in mechanistic/disease models.

Evidence of target engagement can be investigated in vivo using modelsof induced senescence. In order to understand whether test compoundsaccess the disease relevant cell types in the eye, several methods ofsenescence induction can be pursued. DNA damaging agents such asdoxorubicin, bleomycin, and irradiation can induce cellular senescence,and can be directly injected (for example, intravitreal, intracameral,subretinal, etc.) into the eyes of mice (or local or whole-body exposurein the case of irradiation) to drive senescence in the trabecularmeshwork or retina. Test compounds can then be administered to determineaccess of the compounds to appropriate cell layers (as measured by lossof senescence markers). Additionally, many of the SASP factors can bemeasured from these tissues to understand the downstream impact ofsenescence induction, and the impact of senolysis on such mediators.

By way of illustration, administration of bleomycin, a DNA damagingagent, to the anterior chamber of the mouse eye leads to cellularsenescence in the trabecular meshwork (TM), as detected by the inductionof p16 transcript in the TM (Example 8). Elevated relative expression ofp16 mRNA was observed 14 days after intracameral (IC) injection ofbleomycin in the right eye relative to the control left eye.Intracameral administration of a senolytic (UBX1967) on day 7post-bleomycin resulted in a reduction of p16 mRNA on day 14, suggestingclearance of senescent cells by UBX1967 in the mouse TM.

The oxygen-induced retinopathy (OIR) model (Scott and Fruttiger, Eye(2010) 24, 416-421, Oubaha et al, 2016) mimics elements of ischemicretinopathies in humans, such as diabetic retinopathy (DR), retinopathyof prematurity (ROP), and diabetic macular edema (DME). Exposure ofyoung mice to a hyperoxic environment leads to obliteration of retinalvasculature, followed by pathological angiogenesis (neovascularization)upon return to ambient air.

The examples below show the efficacy of the model compound UBX1967 inthe mouse oxygen-induced retinopathy (OIR) model. Intravitreal (IVT)administration of UBX1967 showed statistically significant improvementin the degree of neovascularization and vaso-obliteration at all doselevels (Example 6A). Additionally, we measured the relative abundance ofseveral transcripts associated with senescence (p16, pai1) and humandisease (VEGF) and found that treatment with UBX1967 resulted in areduction in these transcripts. Senescence-associated β-galactosidase(SA-βGal) activity was also reduced after administration of UBX1967.

The streptozotocin (STZ) rodent model (Feit-Leichman et al, IOVS46:4281-87, 2005) recapitulates features of diabetic retinopathy anddiabetic macular edema through the induction of hyperglycemia via thedirect cytotoxic action of STZ on pancreatic beta cells. Hyperglycemiaoccurs within days following STZ administration and phenotypic aspectsof diabetic retinopathy occur within weeks, with vascular leakage andreduced visual acuity and contrast sensitivity demonstrated in theserodents. This model has thus been widely used for the evaluation oftherapeutic agents in diabetic eye disease. The data provided below showthat UBX1967 improved retinal and choroidal vascular leakage.

Other models of retinal ganglion cell damage can be used in testing thatare relevant to glaucoma, where increased intraocular pressure (IOP) isthought to cause retinal ganglion cell loss and optic nerve damage. Inpreclinical species, increased anterior chamber pressure can result inretinal neuron loss as reported in several established models, includingthe magnetic microbead occlusion (Ito et al., Vis Exp. 2016 (109):53731) and other glaucoma models (Almasieh and Levin, Annu Rev Vis Sci.2017). Additionally, ischemia-reperfusion has been demonstrated to causeretinal injury which may result in cellular senescence. Presence ofretinal senescence in such models can be used to monitor the impact ofsenolysis after intravitreal injection of test compounds.

Routes of Administration

Typically a senolytic of this invention is administered directly to theexterior or interior of the eye of the subject, or to a surroundingtissue. Local administration includes topical administration,administration via syringe and/or administration via an implantabledevice. Included is the treatment of anterior (front of the eye) ocularconditions and posterior (back of the eye) ocular conditions.

An anterior ocular condition is a disease, ailment or condition thataffects or involves an anterior ocular region or site, such as aperiocular muscle, an eyelid or an eye tissue or fluid which is locatedanterior to the posterior wall of the lens capsule or ciliary muscles.An anterior ocular condition primarily affects or involves theconjunctiva, the cornea, the anterior chamber, the iris, the posteriorchamber (behind the iris but in front of the posterior wall of the lenscapsule), the lens or the lens capsule and blood vessels and nerveswhich vascularize or innervate an anterior ocular region or site.Examples include dry eye syndromes, conjunctival diseases,conjunctivitis, corneal diseases, presbyopia, cataract, and refractivedisorders. Glaucoma can also be considered to be an anterior ocularcondition because a clinical goal of glaucoma treatment can be to reducea hypertension of aqueous fluid in the anterior chamber of the eye(i.e., reduce intraocular pressure).

A posterior ocular condition is a disease, ailment or condition whichprimarily affects or involves a posterior ocular region or site such assclera, ciliary body, choroid (in a position posterior to a planethrough the posterior wall of the lens capsule), vitreous, posteriorchamber, retina, retinal pigment epithelium, Bruch's membrane, opticnerve (i.e., the optic disc), and blood vessels and nerves whichvascularize or innervate a posterior ocular region or site. Examplesinclude acute macular neuroretinopathy; choroidal neovascularization;histoplasmosis; infections, such as virus-caused infections;non-exudative age related macular degeneration and exudative age relatedmacular degeneration; edema, such as macular edema, cystoid macularedema and diabetic macular edema; multifocal choroiditis; ocular traumawhich affects a posterior ocular site or location; retinal disorders,such as central retinal vein occlusion, diabetic retinopathy,proliferative vitreoretinopathy (PVR), retinal arterial occlusivedisease, retinal detachment, inflammatory chorio-retinal disease;sympathetic ophthalmia; retinitis pigmentosa, and glaucoma. Glaucoma canbe considered a posterior ocular condition because the therapeutic goalis to prevent the loss of or reduce the occurrence of loss of vision dueto damage to or loss of retinal ganglion cells or optic nerve cells(i.e. neuroprotection).

In some cases, an effective amount of a senolytic is delivered to theanterior chamber of the eye via topical administration. The senolyticmay be instilled in the anterior of the eye using eye drops, ointment orgel (e.g., to the conjunctiva and/or cornea) as is needed to treat,ameliorate, and/or prevent the eye disease of interest. The senolyticagent can be an ophthalmic preparation in the form of eye drops thatcontains an amount of the active agent sufficient to provide for atherapeutically effective concentration at the site of action inside theeye.

Local administration to the front of the eye (e.g., to the conjunctivaand/or cornea) may also be done using a contact lens that carries thesenolytic agent. This can improve the bioavailability and prolong theresidence time of an active agent.

To increase the amount of drug load and to control drug release, acontact lens or hydrogel may include: (i) polymeric hydrogels withcontrolled hydrophilic/hydrophobic copolymer ratio; (ii) hydrogels forinclusion of drugs in a colloidal structure dispersed in the contactlenses; (iii) ligand-containing hydrogels; (iv) molecularly imprintedpolymeric hydrogels; (v) hydrogel with the surface containing multilayerstructure for drugs loading and releasing. Hydrogels are a preferredmaterial of soft contact lenses because of their biocompatibility andtransparent characteristic. A hydrogel contact lens can be used torelease an active agent to the front of the eye in a controlled releaseupon contact with the thin film of tears coating the eye. The contactlens can be worn daily according to a dosing schedule to provides forlocal administration of an effective amount of the senolytic fortreatment of an eye disease (e.g., as described herein). Contact lensdevices include those devices described in U.S. Pat. No. 6,827,996 andU.S. Publication Nos. 2010/0330146 and 2006/0251696.

Local administration to the front of the eye can also be done bysubconjunctival injection. A subconjunctival injection can be used toinject a senolytic to either the subconjunctival space or thesub-Tenon's space. Since the subconjunctival space is more anterior thatthe sub-Tenon's space, subconjunctival injections can have a morepronounced effect on drug delivery to the anterior segment, whilesub-Tenon's injections can have more of an effect the posterior segment.

Local administration to the anterior and posterior segments of the eyecan also be achieved by intraocular injection, e.g., an intracameral orintravitreal injection. An intracameral injection is an injection thatis generally delivered into a chamber in the anterior of the eye (e.g.,in front of the lens). An intravitreal injection is an injectiondelivered into the vitreous chamber in the posterior of the eye (e.g.,behind the lens). Because of the risk of damage to the retina layers andoptic nerve by raising intraocular pressure, a maximum volume of about0.1 mL should be administered by either intracameral or intravitrealinjection. In some cases, an intracameral injection can provideadministration without an increase in intraocular pressure that isassociated with an intravitreal injection. A sudden increase inintraocular pressure can cause discomfort to a patient and place theoptic nerve at risk of damage. Administration via injection is generallyperformed in a manner that minimizes exposure of the eye to pathogens.

Local administration can also be done via an implantable ocular device,placed in the eye, for example, by corneal incision. Ocular devicesinclude stents (e.g., trabecular stent), organo-gel implant, and thosecompositions and devices described in U.S. Pat. Nos. 5,501,856,5,869,079, 5,824,072, 4,997,652, 5,164,188, 5,443,505 and 5,766,242.

U.S. Pat. No. 5,501,856 discloses controlled-release pharmaceuticalpreparations for intraocular implants to be applied to the interior ofthe eye after a surgical operation for disorders in retina/vitreous bodyor for glaucoma. U.S. Pat. No. 5,869,079 discloses combinations ofhydrophilic and hydrophobic entities in a biodegradable sustainedrelease implant, and describes a polylactic acid polyglycolic acid(PLGA) copolymer implant comprising dexamethasone. U.S. Pat. No.5,824,072 discloses implants for introduction into a suprachoroidalspace or an avascular region of the eye, and describes a methylcelluloseimplant comprising dexamethasone. U.S. Pat. Nos. 4,997,652 and 5,164,188disclose biodegradable ocular implants comprising microencapsulateddrugs, and describes implanting microcapsules comprising hydrocortisonesuccinate into the posterior segment of the eye. U.S. Pat. No. 5,164,188discloses encapsulated agents for introduction into the suprachoroid ofthe eye, and describes placing microcapsules and plaques comprisinghydrocortisone into the pars plana. U.S. Pat. Nos. 5,443,505 and5,766,242 discloses implants comprising active agents for introductioninto a suprachoroidal space or an avascular region of the eye, anddescribes placing microcapsules and plaques comprising hydrocortisoneinto the pars plana.

To provide a delayed release drug delivery implant or depot, aninjectable formulation including the subject active agent can beinjected into a subject, which results in the in situ formation of anorganogel implant. Upon contact with bodily fluid, crosslinking agentsbegin to crosslink the organogel to form a more stable matrix thatmodulates the escape of the active agent to the eye of the subject. Insome instances, this method of administration can provide a prolongedrelease period of the active agent. In some cases, an in vivobiodegradable cross-linked matrix is formed that includes a non-aqueousaprotic biocompatible solvent system that is non-miscible with water(Zhou, T, et al., “Journal of Controlled Release 55: 281-295, 1998).

Formulation of Medicaments

An ophthalmic preparation can be prepared by mixing a senolytic agentwith a pharmaceutically acceptable base or carrier and as needed one ormore pharmaceutically acceptable excipients. Ingredients acceptable inan ophthalmic formulation are excipients or carriers that cause littleto no ocular irritation, provide suitable preservation if needed, anddeliver one or more agents in a suitable volume. Examples of a base orcarrier include water; an aqueous solvent such as a polar solvent; apolyalcohol; a vegetable oil; and an oily base. Examples of the base orcarrier for an intraocular injection include water for injection andphysiological saline.

For ophthalmic delivery, a senolytic agent may be combined withacceptable excipients for use in and around the eye, such as asurfactant, preservatives, co-solvents, a flavor or cooling agent, anantiseptic, a bactericide or antibacterial agent, a pH adjusting agent,a tonicity agent, a chelating agent, a buffering agent, a stabilizer, anantioxidant, viscosity enhancers, penetration enhancers, sodium chlorideand a thickening agent. In some cases, a composition for intraocularinjection may contain one or more of a solubilizing agent, a suspendingagent, a tonicity agent, a buffering agent, a soothing agent, astabilizer, and an antiseptic. The ophthalmic composition carrier andexcipients can be combined to form an aqueous, sterile ophthalmicsuspension, solution, or viscous or semi-viscous gels or other types ofsolid or semisolid composition such as an ointment.

Exemplary excipients and additives that can be used include thefollowing. Surfactants: for example, nonionic surfactants such aspolyoxyethylene (hereinafter sometimes referred to as“POE”)-polyoxypropylene (hereinafter sometimes referred to as “POP”)block copolymers (e.g., poloxamer 407, poloxamer 235, poloxamer 188),ethylenediamine POE-POP block copolymer adducts (e.g., poloxamine), POEsorbitan fatty acid esters (e.g., polysorbate 20, polysorbate 60,polysorbate 80 (TO-10 etc.)), POE hydrogenated castor oils (e.g., POE(60) hydrogenated castor oil (HCO-60 etc.)), POE castor oils, POE alkylethers (e.g., polyoxyethylene (9) lauryl ether, polyoxyethylene (20)polyoxypropylene (4) cetyl ether), and polyoxyl stearate; amphotericsurfactants such as glycine-type amphoteric surfactants (e.g., alkyldiaminoethyl glycine, alkyl polyaminoethyl glycine), betaine-typeamphoteric surfactants (e.g., lauryldimethylaminoacetic betaine,imidazolinium betaine); cationic surfactants such as alkyl quaternaryammonium salts (e.g., benzalkonium chloride, benzethonium chloride);etc.

Flavors or cooling agents: for example, camphor, borneol, terpenes(these may be in the d-form, l-form, or dl-form); essential oils such asmentha water, eucalyptus oil, bergamot oil, anethole, eugenol, geraniol,menthol, limonene, mentha oil, peppermint oil, rose oil, etc.

Antiseptics, bactericides, or antibacterial agents: for example,polidronium chloride, alkyldiaminoethylglycine hydrochloride, sodiumbenzoate, ethanol, benzalkonium chloride, benzethonium chloride,chlorhexidine gluconate, chlorobutanol, sorbic acid, potassium sorbate,sodium dehydroacetate, methyl paraoxybenzoate, ethyl paraoxybenzoate,propyl paraoxybenzoate, butyl paraoxybenzoate, oxyquinoline sulfate,phenethyl alcohol, benzyl alcohol, biguanide compounds (in particular,polyhexamethylene biguanide or its hydrochloride etc.), Glokill (RhodiaLtd.), etc.

pH adjusting agents: for example, hydrochloric acid, sodium hydroxide,potassium hydroxide, calcium hydroxide, magnesium hydroxide,triethanolamine, monoethanolamine, diisopropanolamine, sulfuric acid,phosphoric acid.

Tonicity agents: for example, sodium bisulfite, sodium sulfite,potassium chloride, calcium chloride, sodium chloride, magnesiumchloride, potassium acetate, sodium acetate, sodium bicarbonate, sodiumcarbonate, sodium thiosulfate, magnesium sulfate, disodium hydrogenphosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate,glycerin, propylene glycol.

Chelating agents: for example, ascorbic acid, edetic acid tetrasodium,sodium edetate, citric acid. Buffering agents: for example, phosphatebuffering agents; citrate buffering agents such as citric acid andsodium citrate; acetate buffering agents such as acetic acid, potassiumacetate, and sodium acetate; carbonate buffering agents such as sodiumbicarbonate and sodium carbonate; borate buffering agents such as boricacid and borax; amino acid buffering agents such as taurine, asparticacid and its salts (e.g., potassium salts etc.), and ε-aminocaproicacid.

Ophthalmic solution formulations may be prepared by dissolving the agentin a physiologically acceptable isotonic aqueous buffer. Further, theophthalmic solution may include an ophthalmologically acceptablesurfactant to assist in dissolving the agent. Viscosity buildingcompounds, such as hydroxymethyl cellulose, hydroxyethyl cellulose,methylcellulose, polyvinylpyrrolidone may be added to improve theretention of the compound.

Sterile ophthalmic gel formulations may be prepared by suspending theagent in a hydrophilic base prepared from the combination of, forexample, CARBOPOL®-940. VISCOAT® (Alcon Laboratories, Inc., Fort Worth,Tex.) may be used for intraocular injection. Other compositions of thepresent invention may contain penetration enhancing materials such asCREMOPHOR® (Sigma Aldrich, St. Louis, Mo.) and TWEEN® 80(polyoxyethylene sorbitan monolaureate, Sigma Aldrich), in the event theagents of the present invention are less penetrating in the eye.

This invention provides commercial products that are kits that encloseunit doses of one or more of the agents or compositions described inthis disclosure. Such kits typically comprise a pharmaceuticalpreparation in one or more containers. The preparations may be providedas one or more unit doses (either combined or separate). The kit maycontain a device such as a syringe for administration of the agent orcomposition in or around the eye of a subject in need thereof. Theproduct may also contain or be accompanied by an informational packageinsert describing the use and attendant benefits of the drugs intreating the senescent cell associated eye disease, and optionally anappliance or device for delivery of the composition.

Ophthalmic Conditions Suitable for Treatment

Provided in the sections that follow is a discussion of specific eyediseases arranged by broad etiologic category (supra) that arecandidates for treatment with a senolytic agent in accordance with thisinvention. The degree to which a particular ophthalmic condition will beamenable to treatment with a senolytic agent will depend on the degreeand extent senescent cells play a role in disease pathology orsymptomatology. The treatment protocol and patient management are withinthe judgment of the managing clinician. The efficacy of the therapy canbe determined empirically.

TYPE 1: Ischemic or Vascular Conditions.

These conditions are characterized by a restriction in blood supply totissues, causing a deficiency of oxygen and/or essential nutrientsneeded for cellular metabolism to keep tissue functional. Ischemia isgenerally caused by diseases associated with blood vessels, withresultant damage to or dysfunction of tissue. It also includes localdeficiencies that arise in a given part of a body resulting from issuesaffecting blood flow but not the vessel itself (such asvasoconstriction, thrombosis, or embolism).

Examples of ischemic or vascular ocular diseases include diabeticretinopathy, glaucomatous retinopathy, ischemic arteritic opticneuropathies, and vascular diseases characterized by arterial and venousocclusion, retinopathy of prematurity and/or sickle cell retinopathy.

The general approach and objectives of senolytic therapy for ischemic orvascular conditions are based on the following:

Ischemia produces a well-known series of pathophysiologic interactionsin the eye. Senolytic therapy impacts this pathophysiology byamelioration of the multiple known inducers of senescence that populatethe ischemic pathway. The primary insult of an ischemic event triggers acascade that exposes the cells of the affected tissue to inducers ofsenescence that include mitochondrial and DNA damage, oxidative stress,inflammation and lipid peroxidation.

The accumulation of senescent cells and release of their associated SASPhas a negative impact on the tissue microenvironment, both for directlyand indirectly impacted cells. The objective of senolytic therapy forischemic diseases of the eye is to decrease the population of senescentcells present in the impacted region, and decrease the associated SASPfactor impact on surrounding cells. This limits ongoing damage in tissuefollowing an ischemic event, and potentially restore function throughimproved features of the cellular microenvironment.

As an example, the elimination of senescent cells in the setting ofischemia impacts visual function by allowing functioning retinalganglion cells to thrive in a healthier local environment, free of theSASP associated detrimental inflammatory, angiogenic and extracellularmatrix-modifying factors. Ischemic events of the visual system commonlyaffect posterior structures of the eye. The ischemia can be influencedby anterior segment features of certain diseases. Thus, the senolyticagent can be delivered in the anterior compartment, or both the anteriorand posterior compartment.

Underlying Pathophysiology

Retinal ischemia contributes to multiple ocular disorders, and occurssecondary to multiple underlying etiologies. Ischemia has beenimplicated in glaucoma, diabetic retinopathy, retinal and choroidalvascular occlusive disease, retinopathy of prematurity and ischemic andtraumatic optic neuropathies. Retinal and macular edema associated withthese conditions is also secondary to the consequences of the ischemiccascade.

FIG. 1A shows the pathophysiologic interactions in the eye that resultfrom ischemia. All known retinal ischemic paradigms result in loss ofganglion cells, crucial to the maintenance of functional vision (OsborneN et al. 2004). Additionally, neovascularization mediated by multiplegrowth factors such as vascular endothelial growth factor (VEGF), TNFα,TGFβ, FGF, PKC, angiopoietins, PDGF, among others (Ucuzian et al.,2010), is another end stage effect of ischemia and is associated withvision threatening complications of both retinal and choroidalneovascular disease (Campochiaro 2015).

Ischemia secondary to the interruption of retinal blood supply of anyetiology initiates a cascade that can ultimately lead to cell death andis known to involve selective neuronal death, cellular edema andneovascularization. This is outlined in detail in FIG. 1A, whichhighlights the key steps in the cascade which include failure of thesodium-potassium ATPase pump, membrane depolarization, and accumulationof sodium and calcium ions in the cytoplasm with subsequent formation ofdestructive free radicals. This process ultimately leads to cell deathby necrosis or apoptosis (Osborne N et al. 2004). The ischemic pathwayis thus populated with known inducers of senescence, and supports thehypothesis that a senolytic agent can impact the effects of multipleischemia-induced senescent responses.

Glaucoma is another example of an ocular disease that illustrates thisprogression to retinal ganglion cell death, influenced by a range offactors that include ischemia (Choi and Kook, 2015), increased IOP,genetic susceptibility and oxidative stress (Wang et al. 2014), each ofwhich may induce senescence and contribute to the changes in theenvironmental milieu produced by senescent cells and their associatedSASP factors.

FIG. 1B shows the multifactorial pathophysiology of glaucoma, leading toretinal ganglion cell (RGC) cell death (Wang et al. 2014).

Glaucoma

Glaucoma has significant associations with age both in prevalence andpathophysiologic characteristics. The prevalence of glaucoma increasesmarkedly over the age of 60 in Caucasians and over 40 inAfrican-Americans and Hispanics. Age is the most consistent and leastvariable risk factor in glaucoma, including IOP. Age is also associatedwith decreased outflow facility in the eye and associated IOP increase,stiffening of the sclera with subsequent impact on the biomechanicalproperties of the optic nerve, and a diminished population of retinalganglion cells (Caprioli, 2013).

Glaucoma is a form of optic neuropathy (a disorder of the optic nerve)that is associated with an increase in intraocular pressure resultingfrom an inability to relieve pressure in the anterior chamber of the eyecaused by an abnormal buildup of the clear fluid known as “aqueoushumor.” There are two primary types of glaucoma: open-angle glaucoma andangle-closure glaucoma. Glaucoma can be classified on the basis ofpathogenesis as primary glaucoma: primary open-angle glaucoma or primaryangle-closure glaucoma; and secondary glaucoma induced by otherdisorders, including steroid-induced glaucoma, pseudoexfoliation orpigment dispersion glaucoma. In open-angle glaucoma (also referred to aswide-angle glaucoma), the trabecular meshwork drain structure or channelof the eye does not drain fluid produced by the ciliary body as itshould, leading to an increase in intraocular pressure. In angle-closure(or narrow-angle) glaucoma, the eye doesn't drain correctly due to a toonarrow angle between the iris and cornea, which can cause a suddenbuildup of intraocular pressure. The presentation of angle closureglaucoma may be acute or chronic. Angle-closure glaucoma can be linkedto farsightedness and cataracts.

Aqueous humor, which is formed in the ciliary body in the posteriorchamber of the eye at the rate of about 2.5 microliters per minute,enters the anterior chamber through a cleft between the front of thelens and the back of the iris through the pupillary opening in the iris.When the eye is functioning normally, the aqueous humor flows out of theanterior chamber at the same or substantially the same rate it entersand, as result, the pressure in the eye remains within the normal rangeof about 12 to 22 mm Hg. Fluids are relatively incompressible, and thusintraocular pressure is distributed relatively uniformly throughout theeye. Increase in intraocular pressure takes place when an imbalancebetween ciliary body production of aqueous fluid and outflow of thisfluid occurs.

Outflow of aqueous humor from the anterior chamber is by two routes. Aminor amount (about 10%) exits through “uveoscleral drainage” betweenmuscle fibers in the ciliary body. This flow is independent ofintraocular pressure. However, the major route of outflow is through thetrabecular meshwork (TM) into Schlemm's canal and is pressure dependent.When this route becomes impeded, the intraocular pressure can becomeelevated because the inflow of aqueous humor is not balanced until thepressure in the eye rises sufficiently to overcome the impediment tooutflow. Our finding of senescent cells in the TM of patients withglaucoma supports TM dysfunction in the pathophysiology of this disease.In time, this can result in loss of vision, both peripheral and central,and eventually lead to complete blindness via loss of retinal ganglioncell and optic nerve function.

Subjects with open-angle glaucoma and chronic angle-closure glaucoma mayhave no obvious symptoms early in the course of the disease. Visualfield loss can occur at later stages of glaucoma. A subject in need oftreatment for glaucoma may exhibit one or more symptoms including, butnot limited to, elevated intraocular pressure, optic-nerve abnormalitieswith corresponding visual field loss, decreased visual acuity, cornealswelling and a closed drainage angle. In certain instances, a subjectwith fluctuating levels of intraocular pressure may experience hazinessof vision and see haloes around lights. In some cases, the symptoms ofacute angle-closure include the rapid onset of eye pain, headache,nausea, vomiting and visual blurring. The eyes of patients with acuteangle-closure glaucoma can appear red, and the pupil of the eye may belarge and nonreactive to light. In certain cases, the cornea may appearcloudy to the naked eye.

Subjects to be treated according to this invention can be selected basedon a clinical presentation or ophthalmic examination that suggests thepresence of glaucoma, using the diagnostic methods outlined previously.These include slit lamp and gonioscopy, tonometry, optic nerve imagingand examination, fundus photography and OCT methods.

The senolytic agents of this invention can provide for a reduction ofintraocular pressure in the eye of a subject in need of treatment, e.g.,a reduction from an elevated intraocular to a normal intraocularpressure through the elimination of senescent cells and associated SASPin the TM and anterior chamber. Optic nerve damage can be alleviated andinhibited by sufficiently reducing intra-ocular pressure and in somecases, can provide for restoration or improvement of retinal visualcapacity.

In some instances, the senolytic is administered by intracameralinjection into the anterior chamber of the eye, or by intravitrealinjection into a posterior segment, vitreous, or vitreous chamber of theeye. The senolytic agents of this invention may be effective to enhanceaqueous humor outflow thereby reducing intraocular pressure.Intravitreal administration may take advantage of the eye's naturalfluid flow: the vitreous humor delivers the active agent to thetrabecular meshwork and uveoscleral pathway as it flows to the anteriorchamber. This can provide for delivery of the active agent to the siteof action in the anterior segment of the eye. Intravitreal delivery mayalso directly target retinal ganglion cells impacted in glaucoma.

Optionally, a senolytic agent can be administered in conjunction with asurgical method of relieving intraocular pressure: for example,canaloplasty, laser trabeculoplasty, trabeculectomy, and the insertionof shunts or other implanted devices. Generally, surgical interventionsprovide temporary relief from elevated intraocular pressure. Implanteddrainage devices include those described in U.S. Pat. No. 9,468,558,which can be inserted in the eye via an incision and positioned, e.g.,on the sclera posterior to the limbus to facilitate drainage.

Diabetic Retinopathy

The prevalence of diabetes and diabetic retinopathy increases with ageand is the most common cause of blindness in people over the age of 50.It is a multifactorial disorder, with hyperglycemia exerting toxiceffects on cells and inflammatory cytokine implicated in many aspects ofdiabetic eye disease (Lutty 2013).

Diabetic eye disease develops in subjects that have diabetes due tochanges in the cells that line blood vessels and encompasses bothvascular and neural dysfunction. Patients with diabetes often developophthalmic complications, such as corneal abnormalities, glaucoma, irisneovascularization, cataracts and neuropathies. Diabetic retinopathy isa common and potentially serious complication of diabetes.

The duration and severity of hyperglycemia is a factor linked to thedevelopment of diabetic retinopathy. When glucose levels are high, as indiabetes, glucose can cause damage in a number of ways. For example,glucose, or a metabolite of glucose, binds to the amino groups ofproteins, leading to tissue damage. In addition, excess glucose entersthe polyol pathway resulting in accumulations of sorbitol. Sorbitolcannot be metabolized by the cells of the retina and can contribute tohigh intracellular osmotic pressure, intracellular edema, impaireddiffusion, tissue hypoxia, capillary cell damage, and capillaryweakening. Diabetic retinopathy involves thickening of capillarybasement membranes and prevents pericytes from contacting endothelialcells of the capillaries. Loss of pericytes increases leakage of thecapillaries and can lead to a breakdown of the blood-retina barrier.Weakened capillaries can lead to aneurysm formation and further leakage.These effects of hyperglycemia can also impair neuronal functions in theretina. This is an early stage of diabetic retinopathy termednonproliferative diabetic retinopathy.

Diabetic retinopathy is also a degenerative disease of the neuralretina, associated with alterations in neuronal function prior to theonset of clinical vascular disease. Retinal capillaries can becomeoccluded in diabetes causing areas of ischemia in the retina. Thenon-perfused tissue responds by eliciting new blood vessel growth fromexisting vessels (i.e., angiogenesis). These new blood vessels can alsocause loss of sight, a condition called proliferative diabeticretinopathy, since the new blood vessels are fragile and tend to leakblood into the eye. In advanced proliferative diabetic retinopathy, anangiogenic, VEGF-mediated response with retinal neovascularizationensues, placing the eye at further risk for severe visual loss due tothe development of vitreous hemorrhage or traction retinal detachment.Irreversible vascular or neuronal damage is possible without treatment,underscoring the need for early intervention.

Symptoms of diabetic retinopathy include loss of central vision,inability to see colors, blurry vision, floaters, distortion, holes orblack spots in vision. In the initial stages of diabetic retinopathy, insome cases, a subject can be asymptomatic. However, microaneurysms inthe eye can be an early clinical sign of diabetic retinopathy. In somecases, the microaneurysm occurs secondary to capillary wall outpouchingdue to pericyte loss, and can appear as small, red dots in thesuperficial retinal layer.

Diagnostic methods include complete ophthalmic examination as outlinedpreviously, with fluorescein angiography, optical coherence tomographyscanning (OCT) and B-scan ultrasonography commonly used ancillary teststo stage and monitor response to therapy. The severity ofnonproliferative diabetic retinopathy can be assessed by the presence,number and locations of microaneurysms and hemorrhages. Microaneurysmscan appear as pinpoint, hyperfluorescent lesions in early phases of theangiogram and typically leak in the later phases of a fluoresceinangiography test. Proliferative diabetic retinopathy can be assessed inpart via the presence of neovascularization, preretinal hemorrhages,hemorrhage into the vitreous, fibrovascular tissue proliferation, andtraction retinal detachments. OCT can be used to determine the thicknessof the retina and the presence of swelling within the retina, as well asassociated vitreomacular traction.

Further signs and symptoms of diabetic retinopathy include: 1) Dot andblot hemorrhages, which can appear similar to microaneurysms if smalland can occur as microaneurysms rupture in the deeper layers of theretina (inner nuclear and outer plexiform layers); 2) Flame-shapedhemorrhages which are splinter hemorrhages that occur in the moresuperficial nerve fiber layer; 3) Retinal edema and hard exudates whichare caused by the breakdown of the blood-retina barrier, allowingleakage of serum proteins, lipids, and protein from the vessels; 4)Cotton-wool spots which are nerve fiber layer infarctions from occlusionof precapillary arterioles; they are frequently bordered bymicroaneurysms and vascular hyperpermeability; 5) Venous loops andvenous beading which can occur adjacent to areas of nonperfusion; theyreflect increasing retinal ischemia, and their occurrence can be apredictor of progression to proliferative diabetic retinopathy (PDR); 6)Intraretinal microvascular abnormalities which include remodeledcapillary beds without proliferative changes; these can usually be foundon the borders of the nonperfused retina; and 7) Macular edema thatcauses visual impairment.

Other Vascular Eye Diseases

Other vascular eye diseases are exemplified by arterial and/or venousocclusion of the retina and/or optic nerve and retinopathy (e.g.,retinopathy of prematurity and/or sickle cell retinopathy).

Retinal vein occlusion (RVO) is a blockage of the small veins that carryblood away from the retina. Blockage of smaller veins (e.g., branchveins) in the retina can occur in places where retinal arteries thathave been thickened or hardened by atherosclerosis cross over and placepressure on a retinal vein. With blockage, pressure builds up in thecapillaries, leading to hemorrhage and leakage of fluid and blood. Thiscan lead to macular edema with leakage near the macula. Macular ischemiaoccurs when these capillaries, which supply oxygen to the retina,manifest leakage and nonperfusion. Neovascularization, new abnormalblood vessel growth, then occurs, which can result in neovascularglaucoma, vitreous hemorrhage, and, in late or severe cases, retinaldetachment. Visual morbidity and blindness that occurs in RVO can resultfrom events of macular edema, retinal hemorrhage, macular ischemia,and/or neovascular glaucoma.

A related event is central or branch retinal artery occlusion (RAO) thatcan occur when a plaque (e.g., blood clot or fat deposit) obstructs ablood vessel or artery in the eye. This can result in a sudden andpermanent loss of vision, usually in just one eye. In some cases ofcentral RAO, fundoscopic examination of the affected eye can show a paleretina with a cherry red macula (i.e., a cherry red spot) that resultsfrom obstruction of blood flow to the retina from the retinal artery,causing pallor, and continued supply of blood to the choroid from theciliary artery, resulting in a bright red coloration at the thinnestpart of the retina (i.e., macula). In general, this does not developuntil after an embolism, and can resolve within days of the acute event.In some cases, by this time visual loss is permanent and primary opticatrophy has developed. In some cases, where a cilioretinal arterysupplies the macula, a cherry red spot is not observed. Branch RAO canoccur when the plaque lodges in a more distal branch of the retinalartery and can involve the temporal retinal vessels. In some cases, asubject at risk of retinal artery occlusion is identified and treatedusing a senolytic agent before serious occlusion occurs.

Retinopathy of prematurity (ROP) (also referred to as retrolentalfibroplasia (RLF)) is a vascular eye disease caused by fibrovascularproliferation, e.g., disorganized growth of retinal blood vessels, whichmay result in scarring and retinal detachment. Oxygen toxicity andrelative hypoxia can contribute to the development of ROP. Variousstages of ROP disease have been defined (Committee for theClassification of Retinopathy of Prematurity, Arch Ophthalmol. 102(8):1130-1134, 1984.

Sickle cell retinopathy (SCR) can develop in subjects suffering fromsickle cell disease, in some cases, during the second decade of life.The ocular manifestations of sickle cell disease (SCD) result fromvascular occlusion, which may occur in the conjunctiva, iris, retina,and choroid. SCR is triggered by vaso-occlusion of the ocularmicrovasculature, as opposed to diabetic retinopathy which can beassociated with overexposure of the vascular tissues to hyperglycemia.SCR may lead to visual impairment depending on its localization andaffected tissue. SCR can be classified as non-proliferative orproliferative according to the presence or absence of neovascularizationin the eye.

In the non-proliferative form of SCR clinical findings can includesalmon patch hemorrhages, iridescent spots and black sunbursts, whichcan be observed in the peripheral retina. Venous tortuosity, enlargementof the foveal avascular zone, central retinal artery obstruction andperipapillary and peri-macular arteriolar occlusions can also beobserved in the central part of the retina.

Proliferative SCR complications can lead to visual impairment or loss in10-20% of affected eyes. In some cases, a subject may be diagnosed by ahistory of spontaneous regression after an initial development ofproliferative SCR. Peripheral retinal neovascularization can developafter vaso-occlusion of the peripheral retina that can grow anteriorlyfrom perfused to non-perfused retina. Initially these new vessels areflat and resemble sea fans. Neovascularization is capable of causingvitreous hemorrhage due to the constant leaking of blood components intothe vitreous through the fragile neovascular tissue. The repetition ofthis hemorrhagic phenomenon leads to worsening of the vitreo-retinaltraction, with the potential of causing rhegmatogenous or tractionalretinal detachment.

Senolytic therapy can be applied to a range of ischemic ocular diseasephenotypes that include RGC loss in glaucoma, diabetic and vascularocclusive retinopathies (e.g. arterial and venous occlusion, retinopathyof prematurity, sickle cell retinopathy, inflammatory and infectiousretinopathies, radiation retinopathy, etc.) and neovascular AMD (knownto be mediated by VEGF and other known SASP factors such as IL-6). Thesephenotypes have well defined clinical features that can be diagnosed andmonitored by a variety of clinical testing procedures includingexamination and tests of retinal structure (for example, fluoresceinangiography for areas of non-perfusion, optical coherence tomography forretinal cellular layer structure, fluid presence, thickness) andfunction (visual field testing, electrophysiology which can specificallymeasure function of the retinal ganglion cells).

In support of this invention, the images in FIGS. 12A and 12B show thepresence of senescent cells in trabecular meshwork tissue of patientswith glaucoma. There is an association of senescent cells with retinalganglion cell layer loss in glaucoma (Skowronska-Krawcyzyk, 2015; Li etal 2017).

TYPE 2: Degenerative Conditions.

These conditions are characterized by a progressive deterioration inquality, function, or structure of the eye, leading to a progressivedecrease in visual acuity.

Examples of degenerative ocular diseases include dermatochalasis,ptosis, keratitis sicca, Fuch's corneal dystrophy, presbyopia, cataract,wet age related macular degeneration (wet AMD), dry age related maculardegeneration (dry AMD); degenerative vitreous disorders, includingvitreomacular traction (VMT) syndrome, macular hole, epiretinal membrane(ERM), retinal tears, retinal detachment, and proliferativevitreoretinopathy (PVR).

The general approach and objectives of senolytic therapy fordegenerative conditions are based on the following:

Degenerative ocular conditions affect all anatomic locations of thevisual system, and are associated with a pathophysiologic cascade thatis populated with features of senescence and SASP-related factorsinduced by a multitude of cellular stressors. These includeenvironmental factors such as UV light exposure and smoking, oxidativestress and inflammatory factors, mitochondrial and DNA damage andextracellular matrix degradation. These stressors are further associatedwith the secretion and accumulation of SASP-like factors which continueto disrupt the local tissue microenvironment and produce ongoingdegenerative changes.

The objective of senolytic therapy is to disrupt this cycle via theelimination of senescent cells and their associated SASP. This wouldlimit ongoing damage in tissue and potentially restore function throughimproved features of the cellular microenvironment. As an example,senolytic therapy can reduce a range of growth factors known to beimplicated in various stages of AMD. A senolytic agent that blocksproduction of SASP factors can exert a multi-pronged impact to thepathophysiologic course of AMD, and can modulate and potentially reversethe course of disease.

Degenerative disorders of the visual system affect anterior andposterior structures therefore the approach to senolytic delivery forthese diseases will include both anterior and posterior deliverymechanisms, depending on the site of the primary pathology.

Dermatochalasis

Dermatochalasis is an age-related change in the upper and lower lidscharacterized by loose, redundant skin and orbicularis muscle often withbulging of the orbital fat pockets. It may be associated with ptosis ofthe eyebrows and forehead relaxation. When severe in the upper lids, itcan limit peripheral vision and obstruct the central visual axis. Thelocalization, severity and frequency of visually significant ptosisincreases with age.

Systemic diseases such as thyroid-related orbitopathy, renal failure,trauma, cutis laxa, Ehlers-Danlos syndrome, amyloidosis, hereditaryangioneurotic edema and xanthelasma may predispose a subject todermatochalasis. In some cases, dermatochalasis is associated with aninherited disorder. Dermatochalasis is caused by a loss of elasticity inthe connective tissue supporting the structure of the front portion ofthe eyelid. The pathophysiology of dermatochalasis is consistent withthe normal aging changes seen in the skin. This includes loss of elasticfibers, thinning of the epidermis, redundancy of the skin, and lymphaticdilation. Subjects with dermatochalasis can also have blepharitis, acondition caused by the plugging of glands in the eye that producelubricating fluid (meibomian glands).

Dermatochalasis can be severe enough that it pushes the eyelashes intothe eye, causing entropion. Eyelid deformities, such as upper eyelidentropion and lower eyelid ectropion or retraction, can be observed withredundant upper or lower eyelid skin. The redundant upper eyelid skinoverhangs the lashes, causing lash ptosis and entropion with resultantkeratitis. In patients with severe lower eyelid dermatochalasis, laxityof the lower eyelid develops with resultant eyelid retraction orectropion.

Dermatochalasis can lead to visual-field loss. In severe cases ofdermatochalasis, a patient can lose 50% or more of their superior visualfield. Patients with a purely aesthetic deformity may not experience anyvisual field defects. Blepharitis is in some cases observed in patientswith moderate-to-severe dermatochalasis. Blepharitis is characterized byeyelid skin edema and erythema; scurf; meibomian gland inflammation andplugging; and, occasionally, hordeolum.

Local administration (e.g., via injection or topical administration tothe eyelid skin and/or muscle) of the senolytic agent can deliver aneffective dose to the subject for the treatment of dermatochalasis. Thesenolytic agents of this invention can halt progression ofdermatochalasis in the subject. In some cases, this invention can haltor reverse at least part of the damage caused prior to treatment.

Ptosis

Ptosis refers to an abnormal position of the eyelid margin, the mostcommon cause of which is aging. Acquired ptosis is a condition wherepatients have normal eyelids when born and develop droopy eyelids inadulthood. The condition can affect one eye or both eyes and is morecommon in the elderly, as muscles in the eyelids may begin todeteriorate. In some cases, a person may be born with congenital ptosisand experience early onset of the condition including drooping of eitherone of both eyelids.

Ptosis occurs due to dysfunction of the muscles that raise the eyelid ortheir nerve supply. Depending upon the cause, ptosis can be classifiedinto several types: Neurogenic ptosis which includes oculomotor nervepalsy, Horner's syndrome, Marcus Gunn jaw winking syndrome, thirdcranial nerve misdirection; Myogenic ptosis which includesoculopharyngeal muscular dystrophy, myasthenia gravis, myotonicdystrophy, ocular myopathy, simple congenital ptosis, blepharophimosissyndrome; Aponeurotic ptosis which may be involutional orpost-operative; Mechanical ptosis which occurs due to edema or tumors ofthe upper lid; Neurotoxic ptosis; and Pseudo ptosis.

The primary symptom of ptosis is a visible drooping or sagging of theupper eyelid, in one or both eyes. Ptosis generally gives the face atired or severe appearance. Ptosis can also result in dry eyes or wateryeyes, as the eyelids are no longer functioning effectively to keep theeyes moist. Severe ptosis can lead to visual field loss. In some cases,the subject experiences tiredness and aching around the eyes, as theeyebrows are constantly lifted in order to see properly.

Local administration can be done via injection or topical administrationto the eyelid skin and/or muscle). In some cases, a senolytic agent canreverse or halt at least part of the damage caused by the condition.

Keratitis Sicca

Keratitis sicca is a condition characterized by dryness of theconjunctiva and/or cornea. Keratitis sicca is also referred to askeratoconjunctivitis sicca, dry eye, dry eye syndrome, dry eye disease(DED) or dysfunctional tear syndrome. Specific subtypes that fall withinthe scope of keratitis sicca include aqueous-deficient DED, evaporativeDED, Sjögren syndrome, lacrimal gland insufficiency and Meibomian glanddisease.

In some cases, keratitis sicca occurs due to inadequate tear productionand may be referred to as aqueous tear-deficient dry eye oraqueous-deficient DED. Aqueous tear-deficient dry eye can be found amongpostmenopausal women or in subjects suffering from an autoimmunedisorder or disease such as rheumatoid arthritis, systemic lupuserythematosus (lupus) or Sjögren syndrome. In certain cases, keratitissicca occurs due to an abnormality of tear composition that results inrapid evaporation of the tears and as such may be referred to asevaporative dry eyes. Drying of the eye can also result from the eyesbeing partly open for periods of time at night (e.g., in a subjectaffected with nocturnal lagophthalmos) or from an insufficient rate ofblinking (e.g., as can occur in subjects with Parkinson's disease).Damage to the surface of the eye that can result from the condition ofkeratitis sicca can include increased discomfort and sensitivity tobright light.

Symptoms of keratitis sicca may include eye irritation, stinging,burning, pain or soreness, itching, a pulling sensation, pressure behindthe eye; a dry, scratchy, gritty, or sandy feeling in the eye; foreignbody sensation, sensitivity to bright light, blurred vision, increasedblinking, eye fatigue, photophobia, redness, mucus discharge, contactlens intolerance and excessive reflex tearing. Foreign body sensationrefers to a sensation or feeling as if something is in the eye. Incertain instances, a subject can experience blurred vision or eyeirritation that is severe, frequent, and/or prolonged in duration. Insome subjects with severe dry eye, the surface of the cornea canthicken, and/or ulcers and scars can develop. In some cases, bloodvessels can grow across the cornea and impair vision.

Not all subjects suffering from keratitis sicca exhibit all symptoms.The subject may complain of an uncomfortable or burning sensation of theeyes. Photophobia or blurred vision may even be present in severe cases.The medical history of the patient may also be suggestive of dry eyes,for example in a patient with a pre-existing diagnosis of acne rosacea,radiation therapy, rheumatoid arthritis, systemic lupus erythematosus,or scleroderma, or other autoimmune disorder. Biomicroscopic examinationwith a slit lamp can be performed to detect meibomitis, conjunctivaldilation, decreased tear meniscus, increased tear debris, mucus strandsor staining patterns consistent with keratitis sicca. A tear breakuptime of less than 10 seconds may also be assessed, and the Schirmer testcan be performed to identify subjects who would benefit from treatment.

The senolytic may be delivered via topical administration to the siteaffected by the keratitis sicca, e.g., to the conjunctiva and/or cornea:for example, via eye drops, ointment, gel or via controlled sustainedrelease from a contact lens installed on the outside of the eye.Delivery via punctal plug systems or direct injection in to themeibomian or lacrimal glands may also be considerations for delivery ofthe senolytic agent. An improvement in the condition may includereduction of inferior corneal fluorescein staining, improvement in aSchirmer test, or improvement in signs and/or symptoms of dry eyesyndrome. Improvements may be measured using the Ocular Surface DiseaseIndex, or the Ocular Comfort Index.

Fuch's Corneal Dystrophy (FCD)

Fuch's corneal dystrophy is a leading cause of corneal transplantationand affects 5% of the US population over the age of 40 years. It ischaracterized by a progressive loss of endothelial cells and cornealdeposition of an abnormal cellular matrix. The cornea progressivelyswells and clouds as remaining endothelial cells are insufficient todehydrate the cornea and maintain its clarity. Increased proteinexpression of known senescence-related genes CDKN1A and CDKN2A have beendemonstrated (Matthaei et al, 2014). Symptoms of FCD include blurredvision and haze and may be associated with pain and corneal blistering.Diagnosis is made through standard ophthalmic tests as outlinedpreviously, and corneal thickness can be measured with cornealpachymetry. Currently available therapies include topical drops andsurgical intervention with partial or total penetrating keratoplasty.Administration of a senolytic agent can be achieved by local delivery tothe cornea or to the anterior chamber adjacent to the cornea in the eye:for example, by intracameral injection, or via an ocular implantabledevice. This may halt progression or reverse at least part of the damagecaused prior to treatment.

Presbyopia

Presbyopia is a condition directly associated with aging, and is nearlyuniversal as middle age is reached. Presbyopia is associated with agingof the eye and results in progressively worsening ability to focusclearly on close objects. It is due to hardening and loss of flexibilityof the lens of the eye causing the eye to focus light behind rather thanon the retina when looking at close objects. Lens hardening can be theresult of decreasing levels of alpha-crystallin in the lens. Weakeningof the ciliary body muscle fibers may also contribute to the inabilityof the ciliary muscle to deform the lens and cause presbyopia. Senolyticadministration may strengthen the ciliary muscle through elimination ofsenescent cells and subsequent decrease in muscle fibrosis that occurswith age.

Symptoms include refractive errors, difficulty reading small print(e.g., in low light conditions), requirement to hold reading materialfarther away, blurring of near objects, eyestrain, headaches andtiredness when performing tasks requiring near vision. Diagnosis can beperformed by an ophthalmologist eye examination including tests forassessing vision and ability to focus on and discern objects.

Administration can be done by local delivery to the lens or to a chamberadjacent to the lens in the eye: for example, by intracameral injection,intravitreal injection, or via an ocular implantable device. This mayhalt progression or reverse at least part of the damage that hasoccurred.

Cataracts

Cataracts remain the largest cause of global blindness, accounting for18 of the 39 million blind individuals worldwide. The incidence is knownto increase with age and there is no region in the world that is immuneto the age-related onset or impact of vision threatening lens opacity.

A cataract is an opacification of the lens of the eye that causes aprogressive, painless loss of vision. Cataracts can be classified bytheir location. A subcapsular cataract occurs at the back of the lens.People with diabetes or those taking high doses of steroid medicationscan have a greater risk of developing a subcapsular cataract. A nuclearcataract forms deep in the nucleus (e.g., a central zone) of the lensand can be associated with aging. A cortical cataract is characterizedby white, wedge-like opacities that start in the periphery of the lensand work their way to the center in a spoke-like fashion. Corticalcataracts can occur in the lens cortex, which surrounds the centralnucleus.

A normal lens inside the eye works much like a camera lens, receivingand focusing light onto the retina for clear vision. The lens alsoadjusts the eye's focus, letting us see things clearly both up close andfar away. The lens is composed primarily of water and protein that isassembled into a highly ordered, interactive macro-structure essentialfor lens transparency and refractive index. Disruption of intra- orinter-protein interactions can alter this structure, exposinghydrophobic surfaces, and causing protein aggregation associated withcataract formation. Over time, a cataract may grow larger and cloud moreof the lens, blocking some light from passing through the lens andscattering the light, preventing crisp focus on the retina.

A number of risk factors are associated with formation of cataracts,including but not limited to, ultraviolet radiation exposure, diabetes,hypertension, obesity, smoking, prolonged use of corticosteroidmedications, statin medicines used to reduce cholesterol, previous eyeinjury or inflammation, previous eye surgery, hormone replacementtherapy, significant alcohol consumption, high myopia, family history.In some cases, cataracts are associated with oxidative disorders. Senilecataract is an age-related, vision-impairing disease characterized bygradual progressive thickening of the lens of the eye. As outlinedthroughout this application, all of these risk factors may be associatedwith the induction of senescence and SASP factors contributing to thedevelopment of disorders of the visual system.

Symptoms that may be associated with cataracts include decreased visualacuity, glare (e.g., seeing halos and starbursts around lights), myopicshift and monocular diplopia. In some cases, the symptoms are exhibitedby one or more of the following: difficulty reading because of aworsening ability to distinguish the contrast between the light and darkof printed letters on a page, needing more light to see well, problemsdistinguishing dark blue from black, blurred vision, colors appearingmore yellow and less vibrant, and mild double vision (also called ghostimages). In certain instances, cataracts can swell and increase thepressure in the eye, causing glaucoma.

A cataract is identified via examination of the eye as outlinedpreviously. Diagnostic methods specific to cataract formationinclude: 1) Examination of the ocular adnexa and intraocular structures,which may provide clues to the patient's cataract etiology, concomitantdisease, and eventual visual prognosis; 2) Swinging flashlight test todetect a Marcus Gunn pupil or a relative afferent pupillary defect(RAPD) indicative of optic nerve lesions or diffuse macular involvementwhich may be alternate causes of decreased vision; 3) Examination ofnuclear size and brunescence (after dilation, nuclear size andbrunescence as indicators of cataract density can be determined prior tophacoemulsification surgery); 4) Direct and indirect ophthalmoscopy—Toevaluate the integrity of the posterior segment; or 5) genetic testingfor a disease that predisposes a subject to development of a cataract.

A senolytic agent may be effective in preventing development ofcataracts, reversing cataracts in a patient, reducing cataract severity,or increasing lens clarity. Administration can include local delivery tothe lens or to a chamber of the eye adjacent to the lens, viaintraocular or intracameral injection, or via an ocular implantabledevice. This can eliminate the need for conventional cataract surgery onthe eye. In general, the method is performed without surgical removal ofthe lens or a portion thereof.

Age-Related Macular Degeneration (AMD)

Macular degeneration refers to a family of diseases that arecharacterized by a progressive loss of central vision associated withabnormalities of Bruch's membrane, the choroid, the neural retina and/orthe retinal pigment epithelium. AMD can be classified into two types:dry macular degeneration and wet macular degeneration. Dry AMD isdefined by the gradual loss of retinal pigment epithelial (RPE) andphotoreceptor cells in the macula. Wet AMD is characterized by thegrowth of abnormal blood vessels beneath the macular epithelium.Age-related developmental changes in retinal morphology and energymetabolism, as well as cumulative effects of environmental exposures mayrender the neural and vascular retina and retinal pigment epitheliummore susceptible to damage in late adulthood. The pathogenesis ofage-related macular degeneration may be related to different processesassociated with aging, including oxidative stress, mitochondrialdysfunction, and inflammatory processes.

Dry AMD (also referred to as nonexudative AMD) is a broad designation,encompassing all forms of AMD that are not neovascular and includingearly and intermediate forms of AMD, and an advanced form of dry AMDknown as geographic atrophy or atrophic AMD. In geographic atrophy,progressive and irreversible loss of retinal cells leads to a loss ofvisual function. AMD-like pathology is characterized by a progressiveaccumulation of characteristic yellow deposits, called drusen (e.g., abuildup of extracellular proteins and lipids), in the macula, betweenthe retinal pigment epithelium and the underlying choroid which isbelieved to damage the retina over time.

Dry AMD patients can have minimal symptoms in earlier stages of thedisease. In approximately 10-20% of subjects, dry AMD progresses to thewet type of AMD. Patients who are affected by dry AMD have gradual lossof central vision due to the death of photoreceptor cells and theirclose associates, retinal pigmented epithelial (RPE) cells, withdeposition of drusen. Photoreceptors, the cells in the retina thatactually ‘see’ light, are essential for vision. RPE cells are necessaryfor photoreceptor survival, function and renewal. The RPE cells in theeye act as macrophages, which phagocytize and recycle components of themembranous outer segments of photoreceptors. If the mitochondria withinthe RPE cells are damaged, photoreceptor recycling is inhibited, withresultant accumulation of drusen. Drusen accumulation causes physicaldisplacement of the RPE from its immediate vascular supply, thechoriocapillaris. This displacement creates a physical barrier that mayimpede normal metabolite and cellular waste diffusion between thechoriocapillaris and the retina.

Symptoms of dry AMD include drusen, visual distortion, e.g., straightlines appear bent, reduced central vision in one or both eyes, need forbrighter light when reading, loss of contrast sensitivity, increasedblurriness of printed words, decreased intensity or brightness ofcolors, difficulty recognizing faces.

The “wet” form of advanced AMD (wet AMD), also referred to asneovascular or exudative AMD, results in vision loss due to abnormalblood vessel growth (e.g., choroidal neovascularization) in thechoriocapillaris, through Bruch's membrane. Wet AMD is preceded by thedry form of AMD and both forms commonly co-exist in the sameindividual—thus both processes may be impacted by simultaneous treatmentwith a senolytic agent. As the photoreceptor and RPE cells slowlydegenerate and breaks from in Bruch's membrane, there is a tendency forblood vessels to grow from their normal location in the choroid into anabnormal location beneath the retina. Choroidal neovascularization (CNV)is stimulated by vascular endothelial growth factor (VEGF). The abnormalblood vessels can be fragile, ultimately leading to blood and proteinleakage below the macula. Bleeding, leaking and scarring from theseblood vessels eventually causes irreversible damage (e.g., hemorrhage,swelling and scar tissue) and severe loss of central vision to thephotoreceptors and rapid vision loss. If left untreated, wet AMD isresponsible for approximately 90% of all blindness resulting from AMD.

Symptoms of wet AMD include reduced central vision in one or both eyes,e.g., dark spot (or spots) in the center of their vision, normal side orperipheral vision, straight lines appearing bent, decreased intensity orbrightness of colors, a general haziness in the overall vision,well-defined blurry or bling spot in field of vision, abnormalneovascularization, blood leakage in the eye, e.g., behind the maculaand abrupt onset or rapid worsening of symptoms.

Eligible subjects may be identified via visual examination of thesubject's eye or according to risk-factor criteria. Testing may includeFundus photography, dark adaptation testing, a Pelli Robson test, visualperformance tests (e.g., Snellen chart, Amsler grid, Farnsworth-Munsell100 hue test and Maximum Color Contrast Sensitivity test), preferentialhyperacuity perimetry test, electroretinography, optical coherencetomography (OCT), and angiography (e.g., fluorescein angiography) in anycombination.

AMD can be characterized into three stages: early, intermediate, andlate, based in part on the extent (size and number) of drusen. Early AMDis diagnosed based on the presence of medium-sized drusen, and may beasymptomatic. Intermediate AMD is diagnosed by large drusen and/or anyretinal pigment abnormalities. In some cases, intermediate AMD may causesome vision loss, however, it is also usually asymptomatic. In late AMD,enough retinal damage occurs that people have symptomatic central visionloss in addition to drusen. The risk of developing symptoms is higherwhen the drusen are large and numerous and associated with disturbancein the pigmented cell layer under the macula. Large and soft drusen arethought to be related to elevated cholesterol deposits.

Diagnosis of macular degeneration rests on signs in the macula,irrespective of visual acuity. A variety of procedures and tests can beused in the diagnosis of AMD as outlined previously.

Relevant drusen features used include number, en face area and volume ofdrusen detected; shape of drusen detected; density of drusen; andreflectivity of drusen. Retinal image data obtained usingspectral-domain optical coherence tomography (SD-OCT) can be analyzed.The image data comprise a cross-section of the retina and an en faceimage of the retina. The image data are processed to obtain an accuratestructure showing locations, shape, size, and other data on drusen. Thisstructural information can provide quantitative drusen features that areindicative of a risk of progression of AMD from the dry form to the wetform of the disease in a given subject and defined time period (U.S.Pat. No. 9,737,205).

Optical coherence tomography can also be used in the diagnosis and thefollow-up evaluation of the response to treatment. In wet AMD,angiography (e.g., fluorescein angiography) allows for theidentification and localization of abnormal vascular processes and canbe used to visualize the leakage of blood behind the macula.

Senolytic therapy can be applied to degenerative retinal diseases suchas age-related macular degeneration (AMD) as follows. Deposits ofabnormal compounds such as lipofuscin in the retinal pigment epithelium(RPE) lead to ongoing accumulation of debris that impacts support of thephotoreceptors in the outer retina. In AMD, this occurs secondary toage-related changes in Bruch's membrane, choriocapillaris and retinalpigment epithelium (RPE). As in ischemia-related ocular disease, acomplex pathophysiologic cascade is initiated, again with multipleavenues for senolytic therapy to impact disease stabilization andpotential regression based on the ability to eliminate senescent cellsand their associated SASP.

FIG. 1C (adapted from Kumar and Fu, 2014) shows factors that influencemultiple points along the AMD pathophysiologic cascade. The presence ofsenescent cells in AMD is demonstrated in FIGS. 14A and 14B. FIG. 8shows the impact of a senolytic agent in senescent RPE cells inaccordance with this invention. The potential for the impact of asenolytic therapy on multiple features of both wet and dry AMD is newand unprecedented.

Degenerative Disorders of the Vitreous:

The vitreous body is a clear gel-like substance that fills the cavity ofthe posterior portion of the eye behind the lens. It is composed of anetwork of collagen fibrils and helps to stabilize the various retinallayers and retinal vasculature. Over time the vitreous gel collapsessecondary to degradation of the collagen fiber network and this putspatients at risk for tractional forces to develop on the underlyingretina. These forces are associated with numerous vitreo-retinaldiseases, all of which increase with age and syneresis of the vitreouscavity which is known to occur in 70% of patients by the age of 70years. These vitreo-retinal conditions are outlined below.

Vitreomacular Traction Syndrome

Vitreomacular traction syndrome (VMT) is a disorder of thevitreo-retinal interface characterized by: (i) an incomplete posteriorvitreous detachment (PVD), (ii) an abnormally strong adherence of theposterior hyaloid face to the macula; and/or (iii) anteroposteriortraction exerted by the syneretic vitreous pulling at adherent sites onthe macula causing morphologic and often functional effects. With age,the vitreous gel undergoes liquefaction forming pockets of fluid withinthe vitreous which leads to a contraction or condensation (syneresis) ofthe vitreous. With loss of vitreous volume, there is a tractional pullexerted at sites of vitreoretinal and vitreopapillary attachments bymeans of the condensing dense vitreous cortex. At the same time, therecan be a weakening of these attachments between the vitreous and theinternal limiting membrane (ILM) leading to detachment of the posteriorhyaloid. Symptoms include blurred or reduced vision, metamorphopsia,micropsia, scotoma, and difficulties with daily vision-related taskssuch as reading.

Macular Hole

Macular hole is a retinal break that in general involves the fovea.Macular holes are related to aging and usually occur in people over age60. Macular holes may be caused by tangential traction as well asanterior posterior traction of the posterior hyaloid on the parafovea.Macular holes can occur as a complication of a posterior vitreousdetachment at its earliest stages. Risk factors for macular hole includeage, myopia, trauma, and ocular inflammation.

Macular holes can begin gradually and develop in three stages: Fovealdetachments (Stage I), which can progress without treatment;Partial-thickness holes (Stage II), which can progress withouttreatment; and Full-thickness holes (Stage III). The size of the holeand its location on the retina can determine how much it will affectvision. When a Stage III macular hole develops, most central anddetailed vision can be lost. If left untreated, a macular hole can leadto a detached retina, a sight-threatening condition.

In the early stage of a macular hole, a subject may notice a slightdistortion or blurriness in their central vision. Straight lines orobjects can begin to look bent or wavy. Reading and performing otherroutine tasks with the affected eye become difficult. Symptoms ofmacular holes include, blurred vision, distorted central vision.

Epiretinal Membrane

Epiretinal membrane (ERM, also referred to as cellophane maculopathy ormacular pucker) is an avascular, fibrocellular tissue that can developon the inner surface of the retina. There are multiple cytokinesassociated with the development and proliferation of ERMs, and anincrease of this condition with age and vitreous syneresis.

The tissue is semi-translucent and proliferates on the surface of theinternal limiting membrane. In the development of ERM, residual corticalvitreous secondary to a posterior vitreous detachment or partialseparation of the posterior hyaloid can allow for the proliferation ofglial cells on the retina. In some cases, ERM is an idiopathiccondition. In certain cases, inflammatory mediators promotefibrocellular growth in the setting of secondary ERM formation.Secondary ERMs can occur in association with retinal vascular diseases,ocular inflammatory disease, trauma, intraocular surgery, intraoculartumors, and retinal tear or detachment. Other risk factors include age,posterior vitreous detachment, and history of ERM in the fellow eye.Symptoms include painless loss of vision, metamorphopsia (visualdistortion, e.g., in which shapes can appears wavy or crooked), doublevision, light sensitivity and images appearing larger or smaller thannormal.

In some cases, subjects with ERMs have few symptoms and their ERMs arediagnosed incidentally on dilated retinal exam or on retinal imagingsuch as with ocular coherence tomography (OCT). OCT imaging method canbe used to assess the severity of the ERM. Fluorescein angiography canalso be used to determine if other underlying retinal problems havecaused the ERM.

Retinal Tear and Detachment

Retinal tears can occur as a result of vitreous traction to an area ofpreexisting vitreoretinal adhesion, most commonly associated with agerelated vitreous syneresis. In some cases, the retina frequentlyappearing completely normal before the acute tear event. A retinal tearmay occur with or without subsequent retinal detachment. Vitreous humorfills the space in the eye between the lens and the retina. Usually,vitreous humor moves from the retina without causing problems, but insome cases, the movement pulls hard enough to tear the retina in one ormore places. Fluid may pass through a retinal tear, lifting or detachingthe retina from the back of the eye.

Retinal detachment occurs when subretinal fluid accumulates between theneurosensory retina and the retinal pigment epithelium. This process canoccur in three ways. One mechanism involves the occurrence of a break inthe retina that allows vitreous to directly enter the subretinal spaceand is known as a rhegmatogenous retinal detachment. Rhegmatogenousretinal detachments are often related to retinal tears associated withposterior vitreous detachment or trauma. A second mechanism involvesproliferative membranes on the surface of the retina or vitreous. Thesemembranes can pull on the neurosensory retina causing a physicalseparation between the neurosensory retina and retinal pigmentepithelium, called a traction retinal detachment.

Tractional retinal detachments can be seen in proliferative retinopathydue to diabetic disease, sickle cell and other disease processes leadingto neovascularization of the retina. Tractional retinal detachments canalso be due to proliferative vitreoretinopathy after trauma or surgery.The third mechanism for retinal detachment is based on accumulation ofsubretinal fluid due to inflammatory mediators or exudation of fluidfrom a mass lesion. This mechanism is known as exudative or serousretinal detachment and can be caused by a number of inflammatory, orexudative retinal disease processes such as sarcoidosis or choroidalneoplasms. Rhegmatogenous retinal detachment has a characteristicappearance differentiating it from a tractional or serous detachment. Arhegmatogenous retinal detachment has a corrugated appearance andundulates with eye movements. Tractional detachments have smooth concavesurfaces with minimal shifting with eye movements. Serous detachmentsshow a smooth retinal surface and shifting fluid depending on patientpositioning.

Retinal tears and rhegmatogenous and tractional detachments requirerepair via any or all of laser, cryotherapy, pneumatic retinopexy,scleral buckle and/or vitrectomy.

Proliferative Vitreoretinopathy

Proliferative vitreoretinopathy (PVR) occurs when a scar forms under oron the retina after retinal detachment, preventing the retina fromhealing and falling back into place. PVR is associated with failedrepair of retinal detachment. When there is a hole in the retina, cellsthat normally reside under the retina enter the eyeball and settle onthe inner layer of the eye on top of the retina. These cells multiplyand form a scar on the surface (and in some cases under) the retina.This scar tissue then contracts and detaches the retina away from theinnermost walls of the eye, resulting in a second retinal detachment.

The senolytic agents of this invention can be administered byintravitreal injection into the vitreous of the eye, or by topicaladministration.

TYPE 3: Genetic Conditions

Genetic ophthalmic conditions are characterized as a disease that iscaused by a mutation, deletion, or insertion in an individual's DNAsequence.

Genetic disorders can be grouped into three main categories: 1. Singlegene disorders: disorders caused by defects in one particular gene,often with simple and predictable inheritance patterns such as dominant,recessive and x-linked. 2. Chromosome disorders: disorders resultingfrom changes in the number or structure of the chromosomes. 3.Multifactorial disorders (complex diseases): disorders caused by changesin multiple genes, often in a complex interaction with environmental andlifestyle factors such as diet or cigarette smoke.

Examples of genetic ocular diseases include Retinitis Pigmentosa,Stargardt Disease, Best Disease and Leber's hereditary optic neuropathy(LHON).

The general approach and objectives of senolytic therapy for geneticconditions are based on the following:

Genetic disorders of the visual system can affect all ocular anatomiclayers, and are associated with cellular defects that may lead to anaccelerated aging phenotype, caused or mediated at least in part bysenescent cells. An inheritable susceptibility to certain eye diseasessuggests that the accumulation of disease-mediating senescent cells maydirectly or indirectly be influenced by genetic components, which againmay lead to earlier presentation. Genetic disorders demonstrate amultifactorial cascade with senescent cells and SASP productioncontributing to ongoing cell dysfunction and degeneration/death.

These disorders can benefit from senolytic therapy because senescentcells and their associated SASP factors mediate associated contributionsto ongoing cell dysfunction, cell loss, and disease progression viablockage of the angiogenic, inflammatory, fibrotic, and extracellularmatrix-modifying proteins present in the pathophysiology. Geneticdisorders of the visual system affect anterior and posterior structures.A senolytic agent is delivered either into the anterior or posteriorregion of the eye, or a combination thereof, depending on the site ofthe primary pathology.

Underlying Pathophysiology

Complex monogenic retinal disorders such as retinitis pigmentosa,Stargardt disease, or disorders of mitochondrial DNA such as Leber'sHereditary Optic Neuropathy, represent diseases with a known geneticbasis that may accelerate the accumulation of senescent cells.Diagnosis, clinical monitoring and response to therapy is monitored inthese diseases as in ischemic or degenerative diseases, by a combinationof functional and structural tests including visual acuity, visualfield, intravenous fluorescein angiogram (IVFA), OCT scanning, andelectrophysiology including electroretinography (ERG) and visual evokedpotential (VEP).

FIG. 1D shows events leading to cell degeneration and cell death inLeber's Hereditary Optic Neuropathy (Mann et al. 2002). FIG. 2A showsretinal cell loss in the clinical stages of Retinitis Pigmentosa (Dryja,1986). These disorders can benefit from the application of a senolyticvia senescent cell and SASP elimination.

In Stargardt disease, lipofuscin accumulation occurs secondary to amutation in the ABCA4 gene. Diagnosis, clinical monitoring and responseto therapy is monitored in these diseases as in ischemic diseases, by acombination of functional and structural tests including visual acuity,visual field, intravenous fluorescein angiogram (IVFA), OCT scanning,and electrophysiology including electroretinography (ERG) and visualevoked potential (VEP).

Retinitis Pigmentosa

Retinitis pigmentosa is a group of inherited disorders that results fromharmful changes in any one of more than 200 genes and leads todegeneration of the retina involving a breakdown and loss of cells inthe retina. Retinitis pigmentosa is characterized by progressiveperipheral vision loss and night vision difficulties (nyctalopia) thatcan lead to central vision loss. In general, the result of the inheritedgene is damage to the photoreceptor cells within the retina. Symptoms ofretinitis pigmentosa include nyctalopia, visual loss, e.g., peripheralvision loss, tunnel vision and photopsia (light flashes).

Variability exists in the physical symptoms depending on the particularform of retinitis pigmentosa and not all subjects exhibit all symptomssimultaneously. Diagnostic methods include visual field testing,fundoscopic examination, electroretinogram (ERG) (measures theelectrical activity of photoreceptor cells) and genetic testing. Ocularexamination can involve assessment of visual acuity and pupillaryreaction, as well as anterior segment, retinal, and fundoscopicevaluation. Subjects with retinitis pigmentosa can have a decreasedelectrical activity, reflecting the declining function ofphotoreceptors. Additional methods that find use in diagnosing otherrelated diseases can be used to eliminate possible disease diagnoses.

In some cases, a DNA sample from the subject can be used for a geneticdiagnosis of retinitis pigmentosa. Particular forms of the disorder canbe genetically typed. Genetic testing is available through the NationalOphthalmic Disorder Genotyping and Phenotyping Network (eyeGENE).

Stargardt Disease

Stargardt disease (also referred to as Stargardt macular dystrophy,juvenile macular degeneration and fundus flavimaculatus) is an inheriteddisorder of the retina. Stargardt disease is one of several geneticdisorders that cause macular degeneration. Stargardt disease ischaracterized by excessive lipofuscin accumulation in the retina whichcauses progressive damage or degeneration of the macula, a small centralarea of the retina responsible for sharp, central vision.

Vitamin A is used to make light-sensitive molecules insidephotoreceptors. Mutations in a gene called ABCA4 are the most commoncause of Stargardt disease. This gene makes a protein that normallyclears away potentially harmful vitamin A byproducts insidephotoreceptors. Affected cells accumulate clumps of lipofuscin, a fattysubstance that forms yellowish flecks. As the clumps of lipofuscinincrease in and around the macula, central vision can become impaired.Eventually, these fatty deposits lead to the death of photoreceptors andvision becomes further impaired.

Symptoms of Stargardt disease include gray, black, or hazy spots in thecenter of their vision; lipofuscin deposits; color blindness, brightlight sensitivity, long adjustment times moving from light to darkenvironments. The progression of symptoms in Stargardt disease can varyfor each subject. Subjects with an earlier disease onset generally havemore rapid vision loss. Vision loss may decrease slowly at first, thenworsen rapidly until it levels off. Subjects with Stargardt disease canend up with 20/200 vision or worse. People with Stargardt disease mayalso begin to lose some of their peripheral vision as they get older.

Diagnostic methods that can be used to assess symptoms of vision loss inStargardt disease include examination and ancillary testing as outlinedpreviously and include visual field testing, color testing, fundusexamination, electroretinography (ERG), optical coherence tomography(OCT) and genetic testing. The number, size, color, and appearance oflipofuscin deposits can be variable from subject to subject. Thelipofuscin deposits can be observed as yellowish flecks in the macula.The flecks are generally irregular in shape and extend outward from themacula in a ring-like pattern.

Accumulation of lipofuscin may be a direct cause of RPE andphotoreceptor demise in the retina. The senolytic agents of thisinvention may preserve vision in Stargardt disease patients affected byexcessive accumulation of lipofuscin.

Best Disease

Best disease (also referred to as vitelliform macular dystrophy) is aform of early onset macular degeneration that is characterized by theappearance of lipofuscin deposits, e.g., in the retinal pigmentepithelium layer of the macula. The lipofuscin deposits can appear asyellow or orange yolk-like or egg-like lesions. Abnormalities in the eyeresult from a disorder in the retinal pigment epithelium (RPE). Adysfunction of the protein bestrophin results in abnormal fluid and iontransport by the RPE. Lipofuscin can accumulate within the RPE cells andin the sub-RPE space, particularly in the foveal area. The RPE of asubject can degenerate over time and in some cases, lead to secondaryloss of photoreceptor cells. In some cases, a breakdown of RPE/Bruchsmembrane can lead to development of choroidal neovascularization.

Symptoms of Best disease include bilateral macular large yellow lesionswith egg-like appearance, mild vision loss in the early stages, moderatevision loss in the late stages, choroidal neovascular membrane (CNVM)and sub-retinal hemorrhage that occurs with mild ocular trauma.

Diagnostic methods include those outlined previously such aselectrooculography (EOG), electroretinography (ERG), optical coherencetomography (OCT), angiography (e.g., fluorescein angiography) and fundusautofluorescence imaging. Fundus autofluorescence (FAF) can be used toshow hyperautofluorescence during the earlier vitelliform stages of thedisease. This hyperfluorescence can settle with the pseduohypopyonstage, becomes mottled with areas of hypoautofluorescence during thevitelleruptive stage, and eventually become hypofluorescent during theatrophic stage of the disease. Changes seen with FAF may precede orappear more striking than with ophthalmoscopy.

Treatment with a senolytic agent may halt or reverse the effect oflipofuscin-associated damage.

Leber's Hereditary Optic Neuropathy (LHON)

Leber's hereditary optic neuropathy (LHON) is a mitochondrial geneticdisease characterized by bilateral loss of central vision owing to focaldegeneration of the optic nerve. The onset of visual loss ranges fromage 8 to 60 but occurs mostly between the age of 15 and 30 years.However, visual deterioration can already occur during the first sevenyears of life. The disease predominantly affects males.

Symptoms of a subject suffering from LHON include blurring or cloudingof vision, loss of visual acuity, loss of central vision, impairment ofcolor vision, centrocecal scotomas, temporal pallor of the optic disc,circumpapillary telangiectatic microangiopathy, sparing of pupillarylight responses, swelling of the retinal nerve fiber layer around thedisc (pseudoedema) and optic atrophy. LHON subjects can remainasymptomatic until experiencing blurring or clouding of vision in oneeye, in some cases, the second eye becoming affected with a delay ofabout six to eight weeks. LHON patients can experience a rapid andpainless loss of central vision accompanied by the fading of colorsespecially in the green/red field. Visual acuity can reach levels of20/400, e.g., in a few months. Optic atrophy is a characteristic of thedisease and can occur after about 6 months.

Diagnostic methods include fundoscopic examination, fluoresceinangiography and optical coherence tomography (OCT). At fundusexamination, characteristic signs of the disease include vasculartortuosity of the central retinal vessels, circumpapillarytelangiectatic microangiopathy and swelling of the retinal nerve fiberlayer around the disc. Fluorescein angiogram may be performed to ruleout true optic nerve edema. In some cases, no dye leakage is noted alongthe borders of an otherwise swollen-appearing optic nerve head. OCT ofthe optic nerve may show elevation in initial phases of the disease, oratrophy in later stages of the disease.

TYPE 4: Infectious Ophthalmic Conditions.

These are diseases caused by pathogenic microorganisms, such asbacteria, viruses, parasites or fungi; the diseases can be spread,directly or indirectly, from one person to another.

Infectious ocular diseases can be caused by bacterial agents, fungalagents, or viruses. Etiologic agents include herpes zoster varicella(HZV), herpes simplex, cytomegalovirus, and human immunodeficiency virus(HIV).

The general approach and objectives of senolytic therapy for infectionsconditions can be based upon the following:

Infectious disorders of the visual system impact all anatomic locationsof the eye and are closely associated with aging and senescence.Infectious agents may contribute to the induction of senescence and amultifactorial cascade with senescent cells and SASP productioncontributing to ongoing cell dysfunction. Once present, senescent cellsmay in turn impact the ability to fight infection.

Senescent cells have an impaired ability to control viral replication(Kim et al, 2016), which is in line with the known increasedsusceptibility to infection that occurs with age. Senescence and theability to respond to infectious agents are a category of ocular diseasethat can be significantly impacted by senolytic therapy. Elimination ofsenescent cells and their associated SASP factors can ameliorate damageto the cellular microenvironment. Infectious disorders of the visualsystem affect anterior and posterior structures. Thus, the senolyticagent is administered to the anterior or posterior compartment, or acombination thereof, depending on the site of the primary pathology.

Herpes Simplex-Associated Ophthalmic Disease

A senolytic agent can be used in treating a subject for viral inducedsenescence caused by herpes simplex-associated ophthalmic disease. Theherpes simplex viruses (e.g., herpes simplex 1) are present in mostadults. The viruses in the herpes family are usually located aroundnerve fibers in humans. Herpes viruses can infect the eye to causeinflammation and scarring of the cornea. Herpes of the eye can betransmitted through close contact with an infected person whose virus isactive. Ranging from a simple infection to a condition that can causeblindness, there are several forms of ocular herpetic infection. Herpeskeratitis is a viral corneal infection that generally affects theepithelium of the cornea. Stromal keratitis occurs when the infectiongoes deeper into the layers of the cornea and can lead to scarringand/or loss of vision. Stromal keratitis may be related to a late immuneresponse to the original infection. Iridocyclitis is a serious form ofinfection where the iris and surrounding tissues inside the eye becomeinflamed, causing severe sensitivity to light, blurred vision, pain andred eyes. When infection occurs in the retina or the inside lining ofthe back of the eye, it can be referred to as herpes retinitis.

Symptoms include pain in and around only one eye, headache and fever,redness, rash or sores on the eyelids and around the eyes, especially onthe forehead or tip of the nose, redness of the eye, blurry vision,inflammation or swelling and/or cloudiness of the cornea, feeling ofdirt or grit in the eye, overflowing tears, pain when looking at brightlight. Diagnostic methods include those previously outlined. In somecases, a Tzanck smear or Wright stain test can be used to determinewhether lesions that are present contain herpes-type virus. Viralculture, direct immunofluorescence assay, and/or PCR methods may also beused to confirm the diagnosis of infection with a particular virus.

The senolytic agent can be administered by local topical administrationto the site affected by the infection e.g., to the conjunctiva and/orcornea to treat, ameliorate, and/or prevent the eye disease. This can beachieved via application of a contact lens that delivers the senolyticwithin the patient's eye(s) at the site of action; or via intraocularinjection to a particular site of infection in the eye. Optionally, thesenolytic agent can be administered in combination with an anti-viralagent, which may include oral agents such as acyclovir, famciclovir orvalacyclovir.

Herpes Zoster Ophthalmicus

Herpes varicella zoster virus (HZV) or varicella zoster virus (VZV) is alatent virus of the herpes family usually located around nerve fibers inhumans. It is strongly associated with increasing age. Reactivation ofthe virus in the ophthalmic division of the trigeminal nerve leads tothe ocular disease Herpes Zoster Ophthalmicus (ZHOU).

Symptoms include severe chronic pain, vision loss, dermatomal foreheadrash, painful inflammation of all the tissues of the anterior and, insome cases, posterior structures of the eye, pain in and around only oneeye, headache and fever, redness of the eye, blurry vision, inflammationor swelling and/or cloudiness of the cornea, feeling of dirt or grit inthe eye, overflowing tears, pain when looking at bright light.

Subjects to be treated according to this invention can be selected basedon a clinical presentation or ophthalmic examination that suggests thepresence of HZO. Diagnostic methods include external inspection, visualacuity, visual fields, extra ocular movements, pupillary response,fundoscopy, intraocular pressure, anterior chamber slit lamp exam, andcorneal exam with and without staining. In some cases, a Tzanck smear orWright stain test can be used to determine whether lesions containherpes-type virus. Viral culture, direct immunofluorescence assay,and/or PCR methods may also be used to confirm the diagnosis ofinfection with a particular virus.

A senolytic agent can be administered by local topical administration tothe site affected by the infection e.g., to the conjunctiva and/orcornea, for example using eye drops, via application of a contact lens,or via intraocular injection to a particular site of infection in theeye. A senolytic agent can be administered in combination with a secondagent, such as a corticosteroid or an anti-viral agent, e.g., an agentactive against herpes varicella zoster virus, such as acyclovir,famciclovir or valacyclovir. In some cases, the anti-viral agent isadministered orally.

Cytomegalovirus Retinitis

Cytomegalovirus (CMV)-associated ophthalmic diseases include CMVretinitis. CMV retinitis is a common opportunistic ocular infection seenmost commonly in immune-compromised hosts (HIV, organ transplant) thatleads to inflammation of the retina and can attack the light-sensingcells in the retina, leading to loss of vision, and in some cases,blindness. Following primary infection, CMV establishes latent infectionin myeloid progenitor cells and intermittent viral reactivation fromactivated macrophages or dendritic cells, which is brought under controlby strong virus-specific CD4+ T-cell and CD8+ T-cell responses. In somecases, patients with HIV become more susceptible to CMV-associatedophthalmic disease when their CD4 count drops below 50 cells/μL.Subjects who are HIV-positive or undergoing immunosuppressivechemotherapy can be susceptible to cytomegalovirus-associated ophthalmicdisease.

Symptoms of CMV retinitis include infection in one or both eyes,floaters, flashes, blind spots, blurred vision, loss of peripheralvision and retinal detachment. The diagnosis of CMV retinitis can beconfirmed by PCR amplification of viral DNA from a sample of thesubject. The CD4+ T-lymphocyte count of a sample of the subject can beused to predict and or diagnose the onset of an ocular infection.Patients with low CD4+ T-lymphocyte counts should undergo regularophthalmologic examinations for retinal symptoms of the disease.

A senolytic agent may be administered in the front of the eye using eyedrops, via application of a contact lens, or via intraocular injectionto a particular site of infection in the eye. In some cases, thesenolytic agent is administered in combination with an anti-viral agentsuch as ganciclovir, valganciclovir, foscarnet or cidofovir.

Human Immunodeficiency Virus (HIV)-Associated Ophthalmic Disease

Human immunodeficiency virus (HIV) can affect the eye either directly orindirectly by means of non-infectious microvascular disorders, orvarious opportunistic infections. Ophthalmic manifestations of HIVinfection may involve the anterior or posterior segment of the eye. Theanterior segment can develop tumors of the periocular tissues and avariety of external infections. posterior segments can develop anHIV-associated retinopathy or an opportunistic infection of the retinaand/or choroid. ophthalmic manifestations of HIV infection includemolluscum contagiosum, herpes zoster ophthalmicus, Kaposi's sarcoma,conjunctival squamous cell carcinoma, trichomegaly, dry eye, anterioruveitis, retinal microvasculopathy, CMV Retinitis, acute retinalnecrosis, progressive outer retinal necrosis, toxoplasmosisretinochoroiditis, syphilis retinitis and Candida albicansendophthalmitis.

Symptoms may include loss of visual acuity, retinal damage and a varietyof other symptoms associated with opportunistic infections of interest.Diagnostic methods include those previously outlined.

In some cases, the CD4+ T-lymphocyte count of a sample of the subject isused to predict and/or diagnose the onset of an ocular infection inpatients who are HIV positive. In some cases, for patients withearly-stage HIV disease (CD4 count>300 cells/μL), ocular syndromesassociated with immunosuppression are uncommon.

The senolytic agent can be administered via intravitreal injection to aparticular site of infection in the eye, optionally in combination withan anti-retroviral HIV agent or specific antivirals such asvalgancyclovir given systemically by injection or oral administration.

TYPE 5: Inflammatory Conditions

These are characterized by a localized response elicited by injury,foreign object, or destruction of tissues, which serves to destroy,dilute, or wall off both the injurious agent and the injured tissue viathe production of pro-inflammatory mediators and the recruitment ofimmune system cells. The inflammatory response can be provoked byphysical, chemical, and biologic agents, including mechanical trauma,exposure to excessive amounts of sunlight, x-rays and radioactivematerials, corrosive chemicals, extremes of heat and cold, or byinfectious agents such as bacteria, viruses, and other pathogenicmicroorganisms. Inflammation can be acute or chronic and associated witha known trigger or idiopathic (i.e. not associated with a clear incitingagent). Autoimmune ocular diseases also occur and can be associated withsignificant visual disability.

Examples of idiopathic posterior inflammatory ocular diseases includepunctate choroiditis (PIC), multifocal choroiditis (MIC) and serpiginouschoroidopathy. Inflammation can also play a role in ocular disordersassociated with other etiologies.

The general approach and objectives of senolytic therapy forinflammatory conditions are based upon the following:

Primary and secondary inflammatory ocular conditions affect all anatomiclocations of the visual system, from lids and cornea to optic nerve andvisual pathway and can be acute or chronic. They are associated with apathophysiologic cascade that is densely populated with features ofsenescence and SASP-related factors. The objective of senolytic therapyis to disrupt the inflammatory cycle via the elimination of senescentcells and their associated SASP. This limits ongoing damage in tissueand potentially restore function through improved features of thecellular microenvironment.

Chronic inflammation is a feature of aging and senescence, and ofnumerous diseases associated with aging. It therefore plays a role inall of the categories of disease presented thus far. Inflammatorydisorders of the visual system affect anterior and posterior structures,and are treated accordingly, depending on the site of the primarypathology.

Punctate Choroiditis (PIC)

PIC is an idiopathic inflammatory disorder of the choroid characterizedby multifocal, well-circumscribed, small choroidal lesions, after aninfectious cause has been ruled out. Changes in the choroidalcirculation related to inflammation may contribute to the pathogenesisof PIC. Symptoms of PIC include blurred vision, photopsia, centraland/or peripheral scotomatas and metamorphopsias.

Diagnostic methods include those previously outlined. On fundoscopy,there can be multiple, small, round, yellowish-white punctate lesions,in the absence of signs of intraocular inflammation. SD-OCT can providestructural characteristics of PIC lesions. Autofluorescence images canshow hypofluorescence in cicatricial lesions and autofluorescence inactive lesions.

The senolytic agent can be administered via intravitreal injection, orvia an intravitreal implant in the vitreous of the eye of a subject(e.g., a bioerodible implant). The senolytic agent may be administeredin combination with corticosteroids, such as cortisone, dexamethasone,fluocinolone, hydrocortisone, methylprednisolone, prednisolone,prednisone and triamcinolone; immunosuppressants such as rapamycin;VEGF-A antagonists such as bevacizumab and ranibizumab; and antibiotics.

Multifocal Choroiditis (MFC)

Multifocal choroiditis (MFC) is a chronic inflammatory disordercharacterized by uveitis (inflammation of the middle layer of the eye)and multiple lesions in the choroid. MFC presents most frequently infemale patients with an age range of 6 to 69 years.

Symptoms of MFC include decreased visual acuity, blurry vision,floaters, sensitivity to light, blind spots, eye discomfort, photopsias,perceived flashes of light, photophobia, inflammation in the front,middle and/or back layers of the eye, posterior uveitis, multiplescattered yellow/gray-white spots in the choroid and/or retina. Thelesions can range in size from 50 to 1,000 μm and have a distribution inthe peripapillary region, within the arcades, and in the mid-peripheryof the eye. In some cases, a subject can develop macular andperipapillary choroidal neovascularization or choroidal neovascularmembranes (CNVMs), new blood vessels that can cause more severe visionloss.

Diagnostic methods include previously outlined techniques, as well asblood tests to eliminate possibility of a particular virus-associateddisease.

The senolytic agent can be administered via intravitreal injection, orvia an intravitreal implant. The senolytic agent can be administered incombination with a second active agent, which is administered topicallyor using an intraocular implant or an intraocular injection. Suitablesecond agents include corticosteroids, such as cortisone, dexamethasone,fluocinolone, hydrocortisone, methylprednisolone, prednisolone,prednisone and triamcinolone; immunosuppressants such as rapamycin;VEGF-A antagonists such as bevacizumab and ranibizumab; and antibiotics.

Serpiginous Choroidopathy

Serpiginous choroidopathy (also referred to as geographic choroiditis orhelicoid peripapillary choroidopathy) is an inflammatory eye conditioncharacterized by progressive destruction of the retinal pigmentepithelium (RPE) and choriocapillaris with secondary involvement of theouter retina. Lesions can form in the eye that last from weeks tomonths, commonly recur, and can involve scarring of the eye tissue. Ifthe centrifugally expanding lesions bypass the fovea, the central visualacuity of the subject can be retained. In the macular variant ofserpiginous choroidopathy, patients can initially develop a geographiclesion in the macula causing early and profound visual loss withoutprior peripapillary activity. The condition develops typically insubjects between age 30 and 70 years and affected patients can show anincreased frequency of HLA-B7 and retinal S-antigen associations.

Symptoms of serpiginous choroidopathy include painless unilateral visionloss, blurred vision, metamorphopsia, photopsias, scotomata, centralscotoma, or yellowish-white chorioretinal lesions with a serpentine orpseudopodal appearance radiating centrifugally from the optic disc.

Diagnostic methods include imaging techniques as previously outlined.

Optionally, a senolytic agent can be administered in combination withother agents such as cortisone, dexamethasone, fluocinolone,hydrocortisone, methylprednisolone, prednisolone, prednisone andtriamcinolone; immunosuppressants such as rapamycin; VEGF-A antagonistsif indicated for associated choroidal neovascularization such asbevacizumab and ranibizumab; and antibiotics.

TYPE 6: Iatrogenic Conditions

Iatrogenic ophthalmic conditions are characterized as disease that isthe result of diagnostic and therapeutic procedures undertaken on apatient. Examples of iatrogenic ocular conditions include causedpost-vitrectomy cataract, or radiation retinopathy following treatmentfor a neoplasm.

The general approach and objectives of senolytic therapy for iatrogenicconditions are based on the following:

The induction of senescence by stressors associated with a procedure ortherapeutic agent produces a pathophysiologic cascade similar to thatseen in other forms of senescence induction. Ocular tissues subject tosuch stressors may have a higher prevalence of senescent cells, which inturn may lead to presentation of certain eye diseases at an earlierstage, or in a more severe form. The objective of senolytic therapy isto disrupt the senescent cell impact on tissue via the elimination ofsenescent cells and their associated SASP. This prevents or limitongoing damage in tissue and potentially restores function throughimproved features of the cellular microenvironment. Given that in somecases the iatrogenic effect can be predicted, preventive therapy may beachievable.

Iatrogenic disorders of the visual system affect anterior and posteriorstructures. Thus, the senolytic agent is applied into the anterior orposterior compartment or both, depending on the site of the primarypathology, the nature of the procedure, and the senolytic agent that ischosen for use

Post-Vitrectomy Cataract

Cataract formation or acceleration can occur after intraocular surgery.Vitrectomy is a microsurgical technique used to gain access to thevitreous cavity and retina, and which is used to treat disorders thataffect the posterior segment of the eye. Vitrectomy is associated withco-morbidities that may compromise visual acuity such as retinaldetachment, corneal decompensation, and cataract formation orprogression. In some cases, the type of cataract that forms oraccelerates after vitrectomy is a nuclear sclerotic cataract. In somecases of eyes undergoing vitrectomy, the lens is also removed. Cataractsthat develop after vitrectomy can limit visual acuity outcomes to adegree that would result in surgical removal of the lens in an otherwisenormal eye. Cataract formation or acceleration after vitrectomy may beassociated with light toxicity, oxidation of lens proteins, use ofintraocular gas, length of operative time, and increased retrolentaloxygen levels.

Symptoms of post-vitrectomy cataracts include decreased visual acuitydespite anatomic and/or functional success of a previous vitrectomysurgery, glare, halos, etc. Individuals who have undergone vitrectomymay have lower levels of baseline (pre-cataract) visual acuity due tothe underlying nature of their retinal pathology; therefore patientswith post-vitrectomy cataract are more likely to present with poorervision than individuals with typical senile cataracts.

Radiation Retinopathy

Radiation retinopathy is a radiation dose-dependent complication ofexposure to radiation, such as external beam radiation or plaquebrachytherapy. External beam radiation is used as treatment fornasopharyngeal, paranasal sinus or orbital tumors, where there islimited ability to protect the eye, and can lead to clinicallysignificant radiation retinopathy. Plaque brachytherapy for treatment ofintraocular tumors can also cause damage to the immediate retina andchoroid. Exposure to radiation may cause preferential loss of vascularendothelial cells with relative sparing of the pericytes. Differentialsensitivity between endothelial cells and pericytes may result fromdirect exposure of the endothelial cells to high ambient oxygen and ironfound in the blood which generates free radicals and leads to cellmembrane damage, occlusion of capillary beds and microaneurysmformation. The retinal ischemia from areas of retinal non-perfusionultimately leads to macular edema, microaneurysm neovascularization,macular edema, vitreous hemorrhage and tractional retinal detachment.

Symptoms of radiation retinopathy include decreased vision, floaters,telangiectase, neovascularization, vitreous hemorrhage, hard exudates,cotton wool spots and/or macular edema.

Diagnostic methods include ophthalmic examination of a subject's eye,for example, by angiography and optical coherence tomography (OCT). Afluorescein angiogram can be used to identify and highlight themicrovascular features of radiation retinopathy. Indocyanine greenangiography can reveal precapillary arteriolar occlusion and areas ofchoroidal hypoperfusion. Optical coherence tomography (OCT) can find usein evaluating macular edema.

Combination of Senolytic Agents with Approved Standard-of-Care Therapies

Senolytic agents for treating ophthalmic conditions can be combined withother pharmaceutical agents that are approved for clinical use. Sincethe removal of senescent cells works by a different mechanism fromcurrent therapies, the two agents can operate synergistically oradditively to minimize the administration schedule and improve outcomes.The senolytic agent will remove senolytic cells in the eye that arepromoting persistence and progression of disease-relatedpathophysiology.

The standard of care for many vascular-related ophthalmic conditions iscurrently an agent that inhibits vascular endothelial growth factor(VEGF): namely, aflibercept (marketed as EYLEA® by RegeneronPharmaceuticals). Aflibercept is a recombinant fusion protein consistingof VEGF-binding portions from the extracellular domains of human VEGFreceptors 1 and 2, that are fused to the Fc portion of the human IgG1immunoglobulin.

For neovascular (Wet) Age-Related Macular Degeneration (AMD), therecommended dose for EYLEA is 2 mg (0.05 mL or 50 microliters),administered by intravitreal injection every 4 weeks (monthly) for thefirst 12 weeks (3 months), followed by 2 mg (0.05 mL) via intravitrealinjection once every 8 weeks (2 months). For macular edema followingretinal vein occlusion (RVO), the recommended dose for EYLEA is 2 mg(0.05 mL or 50 microliters) administered by intravitreal injection onceevery 4 weeks (monthly). For diabetic macular edema (DME), therecommended dose for EYLEA is 2 mg (0.05 mL or 50 microliters)administered by intravitreal injection every 4 weeks (monthly) for thefirst 5 injections, followed by 2 mg (0.05 mL) via intravitrealinjection once every 8 weeks (2 months). Although EYLEA may be dosed asfrequently as 2 mg every 4 weeks (monthly), additional efficacy was notdemonstrated in most patients when EYLEA was dosed every 4 weekscompared to every 8 weeks. Some patients may need every 4-week (monthly)dosing after the first 20 weeks (5 months). For diabetic retinopathy(DR) in patients with DME, the recommended dose for EYLEA is 2 mg (0.05mL or 50 microliters) administered by intravitreal injection every 4weeks (monthly) for the first 5 injections, followed by 2 mg (0.05 mL)via intravitreal injection once every 8 weeks (2 months).

Use of a senolytic agent in combination with anti-VEGF therapy is a newparadigm that can impact the disease features in important ways.Anti-VEGF therapy given alone requires frequent administration toprevent progression of disease. Combining anti-VEGF therapy with asenolytic agent may decrease the rate of progression or the rate ofrecurrence of active disease. It is possible that an anti-VEGF canacutely block the potential for leakage and bleeding and the senolyticagent can impact the CNV sub-acutely and then chronically—with thepossibility that the need for combination therapy is reduced orobviated, or that it is used with a greater degree of efficacy in abroader group of patients. This in turn may allow the managing clinicianto administer the anti-VEGF therapy on a less frequent basis, withimproved visual outcomes.

The combination is expected to improve the response rate, improveoutcomes, and reduce the treatment burden and combined cost of therapy.It can also impact aspects of the underlying dry AMD that are notaddressed by anti-VEGF therapy. This is evident in long term outcomes ofpatients with wet AMD treated with anti-VEGF therapy who develop visionloss over time in the absence of active neovascularization, but ratherdue to atrophy or progression of the underlying dry AMD (Bhisitkul etal., 2015).

A senolytic agent can be combined with other agents that address theunderlying pathophysiology or symptomatology of the condition.

Non-pharmaceutical intervention that can be performed in conjunctionwith senolytic administration includes punctal occlusion to decrease theoutflow of tears from the eye. Another example is fitting the subjectwith scleral or semi-scleral contact lenses that create a fluid-filledlayer over the cornea. In some cases, administration of the senolytic isperformed in combination with a punctum plug device that blocks a tearduct draining tears from the eye.

In some cases, the eye condition is associated with other inflammatoryconditions, and the senolytic agent is administered in combination withantihistamines (such as pheniramine, emedastine, or azelastine),decongestants (such as tetrahydrozoline hydrochloride or naphazoline),or a non-steroidal anti-inflammatory agent (such as nepafenac orketorolac), corticosteroids (such as fluorometholone or loteprednol),mast cell stabilizers (such as azelastie, cromal, emedastine, ketotifen,Iodoxamine, nedocromil, olopatadine, or pemirolast), or steroids. Insome cases, the pharmaceutical composition is administered inconjunction with a second active agent, e.g., a macrolideimmunosuppressant such as ciclosporin, tacrolimus, pimecrolimus,everolimus, sirolimus, deforolimus, temsirolimus, and zotarolimus,abetimus, gusperimus, or mycophenolic acid.

If the target eye disease is associated with an infectious bacterialcondition (such as meibomian gland infection or corneal infection) theeye drops or ointment can be administered with or in a combinationcomposition that contains an antibiotic, such as ciprofloxacin,erythromycin, gentamicin, ofloxacin, sulfacetamine, tobramycin, ormonofloxacin. If the dry eye condition is associated with a viralinfection, the senolytic agent can be administered with or in acombination composition with an anti-viral agent such as trifluridine oridoxuridine. In another example, the subject presents with apre-existing autoimmune disorder. The subject can also be treated with asystemic (for example) oral therapy for the associated condition.

In some instances, the senolytic agent is administered in conjunctionwith one or more different biologically active agents which may be ofthe same or different drug classes. Biologically active agents or drugsare selected from: anesthetics and analgesics, antiallergenics,antihistamines, anti-inflammatory agents, anti-cancer agents,antibiotics, antiinfectives, antibacterials, anti-fungal agents,anti-viral agents, cell transport/mobility impending agents,antiglaucoma drugs, antihypertensives, decongestants, immunologicalresponse modifiers, immunosuppressive agents, peptides and proteins,steroidal compounds (steroids), low solubility steroids, carbonicanhydrize inhibitors, diagnostic agents, antiapoptosis agents, genetherapy agents, sequestering agents, reductants, antipermeabilityagents, antisense compounds, antiproliferative agents, antibodies andantibody conjugates, bloodflow enhancers, antiparasitic agents,non-steroidal anti-inflammatory agents, nutrients and vitamins, enzymeinhibitors, antioxidants, anticataract drugs, aldose reductaseinhibitors, cytoprotectants, cytokines, cytokine inhibitors, andcytokine protectants, UV blockers, mast cell stabilizers, and antineovascular agents such as antiangiogenic agents like matrixmetalloprotease inhibitors and Vascular endothelial growth factor (VEGF)modulators, neuroprotectants, miotics and anti-cholinesterase,mydriatics, artificial tear/dry eye therapies, anti-TNFα, IL-1 receptorantagonists, protein kinase C-13 inhibitors, somatostatin analogs andsympathomimetics.

Other combinations included in the invention may be found elsewhere inthis disclosure.

Definitions

A “senescent cell” is generally thought to be derived from a cell typethat typically replicates, but as a result of aging or other event thatcauses a change in cell state, can no longer replicate. It remainsmetabolically active and commonly adopts a senescence associatedsecretory phenotype (SASP) that includes chemokines, cytokines andextracellular matrix and fibrosis modifying proteins and enzymes. Thenucleus of senescent cells is often characterized bysenescence-associated heterochromatin foci and DNA segments withchromatin alterations reinforcing senescence. Without implying anylimitation on the practice of what is claimed in this disclosure that isnot explicitly stated or required, the invention is premised on thehypothesis that senescent cells cause or mediate certain conditionsassociated with tissue damage or aging. For the purpose of practicingaspects of this invention, senescent cells can be identified asexpressing at least one marker selected from p16, senescence-associatedβ-galactosidase, and lipofuscin; sometimes two or more of these markers,and other markers of SASP such as but not limited to interleukin 6, andinflammatory, angiogenic and extracellular matrix modifying proteins.

A “senescence associated” disease, disorder, or condition is aphysiological condition that presents with one or more symptoms orsigns, wherein a subject having the condition needs or would benefitfrom a lessening of such symptoms or signs. The condition is senescenceassociated if it is caused or mediated in part by senescent cells, whichmay be induced by multiple etiologic factors including age, DNA damage,oxidative stress, genetic defects, etc. Lists of senescence associateddisorders that can potentially be treated or managed using the methodsand products taught in this disclosure include those discussed in thisdisclosure and the previous disclosures to which this application claimspriority.

A compound is typically referred to as “senolytic” if it eliminatessenescent cells, compared with replicative cells of the same tissuetype, or quiescent cells lacking SASP markers. Alternatively, or inaddition, a compound or combination may effectively be used according tothis invention if it decreases the release of pathological solublefactors or mediators as part of the senescence associated secretoryphenotype that play a role in the initial presentation or ongoingpathology of a condition, or inhibit its resolution. In this respect,the term “senolytic” is exemplary, with the understanding that compoundsthat work primarily by inhibiting rather than eliminating senescentcells (senescent cell inhibitors) can be used in a similar fashion withensuing benefits.

Successful “treatment” of an eye disease according to this invention mayhave any effect that is beneficial to the subject being treated. Thisincludes decreasing severity, duration, or progression of a condition,or of any adverse signs or symptoms resulting therefrom. In somecircumstances, senolytic agents can also be used to prevent or inhibitpresentation of a condition for which a subject is susceptible, forexample, because of an inherited susceptibility of because of medicalhistory.

A “therapeutically effective amount” is an amount of a compound of thepresent disclosure that (i) treats the particular disease, condition, ordisorder, (ii) attenuates, ameliorates, or eliminates one or moresymptoms of the particular disease, condition, or disorder, (iii)prevents or delays the onset of one or more symptoms of the particulardisease, condition, or disorder described herein, (iv) prevents ordelays progression of the particular disease, condition or disorder, or(v) at least partially reverses damage caused by the condition prior totreatment.

The terms “infectious eye disease” and “ophthalmic infection” refer toan infection caused by a microorganism or microorganisms in or around aneye or the eye structure which include the eyelids and lacrimalapparatus, the conjunctiva, the cornea, the uvea, the vitreous body, theretina, and the optic nerve.

A “phosphorylated” form of a compound is a compound in which one or more—OH or —COOH groups have been substituted with a phosphate group whichis either —OPO₃H₂, —OC_(n)H_(2n)PO₃H₂, or —OC(═O)C_(n)H_(2n)PO₃H₂ (wheren is 1 to 4), such that the phosphate group or groups may be removed invivo (for example, by enzymolysis). A non-phosphorylated ordephosphorylated form has no such phosphate group.

Unless otherwise stated or required, all the compound structuresreferred to in the invention include conjugate acids and bases havingthe same structure, crystalline and amorphous forms of those compounds,pharmaceutically acceptable salts, and dissolved and solid formsthereof, including, for example, polymorphs, solvates, hydrates,unsolvated polymorphs (including anhydrates), conformational polymorphs,and amorphous forms of the compounds, as well as mixtures thereof.

An ophthalmic or ocular disorder is defined for the purposes of thisdisclosure as a disorder of the visual system. An anterior ocularcondition is a disease, ailment or condition that affects or involves ananterior ocular region or site, such as a periocular muscle, an eye lidor an eye tissue or fluid which is located anterior to the posteriorwall of the lens capsule or ciliary muscles. A posterior ocularcondition is a disease, ailment or condition which primarily affects orinvolves a posterior ocular region or site such as choroid or sclera (ina position posterior to a plane through the posterior wall of the lenscapsule), vitreous, vitreous chamber, retina, retinal pigmentedepithelium, Bruch's membrane, optic nerve (i.e. the optic disc), visualpathway and blood vessels and nerves which vascularize or innervate aposterior ocular region or site.

Except where otherwise stated or required, other terms used in thespecification have their ordinary meaning.

INCORPORATION BY REFERENCE

For all purposes in the United States and in other jurisdictions whereeffective, each and every publication and patent document cited in thisdisclosure is hereby incorporated herein by reference in its entiretyfor all purposes to the same extent as if each such publication ordocument was specifically and individually indicated to be incorporatedherein by reference.

US 2016/0339019 A1 (Laberge et al.) and WO 2016127135 (David et al.) arehereby incorporated herein for all purposes, including but not limitedto the identification, formulation, and use of compounds capable ofeliminating or reducing the activity of senescent cells and treatingophthalmic conditions. U.S. Pat. Nos. 8,691,184, 9,096,625, and9,403,856 (Wang et al.) are hereby incorporated herein by reference inits entirety for all purposes, including the features of compounds inthe Bcl library, their preparation and use.

EXAMPLES Example 1: Measuring Bcl Inhibition

The ability of candidate compounds to inhibit Bcl-2 and Bcl-xL activitycan be measured on the molecular level by direct binding. This assayuses a homogenous assay technology based on oxygen channeling that ismarketed by PerkinElmer Inc., Waltham, Mass.: see Eglin et al., CurrentChemical Genomics, 2008, 1, 2-10. The test compound is combined with thetarget Bcl protein and a peptide representing the corresponding cognateligand, labeled with biotin. The mixture is then combined withstreptavidin bearing luminescent donor beads and luminescent acceptorbeads, which proportionally reduces luminescence if the compound hasinhibited the peptide from binding to the Bcl protein.

Bcl-2, Bcl-xL and Bcl-w are available from Sigma-Aldrich Co., St. Louis,Mo. Biotinylated BIM peptide (ligand for Bcl-2) and BAD peptide (ligandfor Bcl-xL) are described in US 2016/0038503 A1. AlphaScreen®Streptavidin donor beads and Anti-6×His AlphaLISA® acceptor beads areavailable from PerkinElmer

To conduct the assay, a 1:4 dilution series of the compound is preparedin DMSO, and then diluted 1:100 in assay buffer. In a 96-well PCR plate,the following are combined in order: 10 μL peptide (120 nM BIM or 60 nMBIM), 10 μL test compound, and 10 μL Bcl protein (0.8 nM Bcl-2/W or 0.4nM Bcl-XL). The assay plate is incubated in the dark at room temperaturefor 24 h. The next day, donor beads and acceptor beads are combined, and5 μL is added to each well. After incubating in the dark for 30 minute,luminescence is measured using a plate reader, and the affinity ordegree of inhibition by each test compound is determined.

Example 2: Measuring MDM2 Inhibition

MDM2 (mouse double minute 2 homolog, also known as E3 ubiquitin-proteinligase) is a negative regulator of the p53 tumor suppressor. InhibitingMDM2 promotes p53 activity, thereby conferring senolytic activity. Theability of compounds to act as agonists for MDM2 can be measuredindirectly in cells by monitoring the effect on p53.

A p53 luciferase reporter RKO stable cell line can be obtained fromSignosis Inc., Santa Clara Calif. In the p53 luciferase cell line,luciferase activity is specifically associated with the activity of p53.The cell line was established by transfection of a p53 luciferasereporter vector along with a G418 expression vector, followed by G418selection.

The assay is conducted as follows. Cells from the reporter cell line aretreated for 24 h with the candidate compound. Media is then removed, thecells are washed with PBS, and 20 μL of lysis buffer is added to eachwell. Cells are shaken for 10 s using a plate reader agitator.Luciferase buffer is prepared and added to the wells. p53 activity isthen read using a Victor™ multilabel plate reader (PerkinElmer, San JoseCalif.).

Example 3: Measuring Senolytic Activity Using Senescent Fibroblasts

Human fibroblast IMR90 cells can be obtained from the American TypeCulture Collection (ATCC®) with the designation CCL-186. The cells aremaintained at <75% confluency in DMEM containing FBS and Pen/Strep in anatmosphere of 3% O₂, 10% CO₂, and ˜95% humidity. The cells are dividedinto three groups: irradiated cells (cultured for 14 days afterirradiation prior to use), proliferating normal cells (cultured at lowdensity for one day prior to use), and quiescent cells (cultured at highdensity for four day prior to use).

On day 0, the irradiated cells are prepared as follows. IMR90 cells arewashed, placed in T175 flasks at a density of 50,000 cells per mL, andirradiated at 10-15 Gy. Following irradiation, the cells are plated at100 μL in 96-well plates. On days 1, 3, 6, 10, and 13, the medium ineach well is aspirated and replaced with fresh medium.

On day 10, the quiescent healthy cells are prepared as follows. IMR90cells are washed, combined with 3 mL of TrypLE trypsin-containingreagent (Thermofisher Scientific, Waltham, Mass.) and cultured for 5 minuntil the cells have rounded up and begin to detach from the plate.Cells are dispersed, counted, and prepared in medium at a concentrationof 50,000 cells per mL. 100 μL of the cells is plated in each well of a96-well plate. Medium is changed on day 13.

On day 13, the proliferating healthy cell population is prepared asfollows. Healthy IMR90 cells are washed, combined with 3 mL of TrypLEand cultured for 5 minutes until the cells have rounded up and begin todetach from the plate. Cells are dispersed, counted, and prepared inmedium at a concentration of 25,000 cells per mL. 100 μL of the cells isplated in each well of a 96-well plate.

On day 14, test Bcl-2 inhibitors or MDM2 inhibitors are combined withthe cells as follows. A DMSO dilution series of each test compound isprepared at 200 times the final desired concentration in a 96-well PCRplate. Immediately before use, the DMSO stocks are diluted 1:200 intoprewarmed complete medium. Medium is aspirated from the cells in eachwell, and 100 μL/well of the compound containing medium is added.

Candidate senolytic agents for testing are cultured with the cells for 6days, replacing the culture medium with fresh medium and the samecompound concentration on day 17. Bcl-2 inhibitors like 001967 arecultured with the cells for 3 days. The assay system uses the propertiesof a thermostable luciferase to enable reaction conditions that generatea stable luminescent signal while simultaneously inhibiting endogenousATPase released during cell lysis. At the end of the culture period, 100μL of CellTiter-Glo® reagent (Promega Corp., Madison, Wis.) is added toeach of the wells. The cell plates are placed for 30 seconds on anorbital shaker, and luminescence is measured.

Example 3A Screening a Compound Library for Bcl Antagonists

Discovery of senolytic agents useful for implementation according tothis invention was based on the premise that senescent cells can bekilled by inhibiting one or more of the Bcl family of regulator proteinsthat are anti-apoptotic. A molecule with high affinity and selectivityfor a Bcl isoform was hypothesized to be effective in inducing apoptosisin senescent cells but not proliferating or non-senescent cells of thesame tissue type. Compounds with these properties would be candidatesfor development as therapeutic agents for clinical medicine.

A library that was initially constructed that contained several hundredcompounds. Synthesis and use of such library are explained in U.S. Pat.Nos. 8,691,184, 9,096,625, and 9,403,856. The library was initiallyscreened elsewhere for compounds that were able to bind or inhibitBcl-xL and/or Bcl-2. Fifteen compounds were chosen from the initialscreening for further analysis. FIGS. 1A, 1B, and 1C show nine of thefifteen compounds.

The chosen compounds were further assayed to quantitatively determinethe actual affinity for Bcl-xL, Bcl-2, and Bcl-w with a view toidentifying candidate senolytic agents for use in treating age relatedconditions. FIGS. 2A, 2B, and 2C show the results of the binding assay,with those compounds towards the left of each graph having the highestaffinity.

Individual compounds in the library had similar core structures. Asshown in FIGS. 3A, 3B, and 3C, the structures of each of the compoundshaving the most promise were compared with a view to identifyingsubstituents of the molecular structure that were contributing to thedesired effect.

Example 3B: Screening High Affinity Bcl Antagonists for SenolyticPotency and Specificity

An ability to bind Bcl regulatory proteins does not necessarily meanthat the compound is suitable for inducing apoptosis in a clinicalsetting. Furthermore, even if the compound is potent, it would not besuitable for use as a therapeutic unless it preferentially killssenescent cells with a high degree of selectivity. Accordingly,compounds in the library showing high Bcl binding affinity were furtherscreened for their ability to kill irradiated fibroblasts, in comparisonwith replicating fibroblasts or fibroblasts that were quiescent (due toconfluence) but not senescent.

FIG. 4 shows data obtained from the nine model compounds: affinity ofbinding to each of the Bcl isoforms, and the effective concentration(EC₅₀) for killing senescent fibroblasts (SnCs) in culture. The data aresummarized in TABLE 1, below.

Binding Affinities (K_(i) ± SD) Best Original Molecular Bcl-xL Bcl-2Bcl-w SnEC₅₀ IDs IDs Weight (pM) (pM) (pM) (μM) SI UM/APG-901 BM-501639.1 NA NA NA NA NA UM/APG-902 BM-983 967.5 3307 4037 >250000 5.000 1.0UM/APG-903 BM-782 1274.4 885.55 10923 17694 11.000 1.3 UM/APG-904 BM-7881148.2 5292 121045 23132.5 NA NA UM/APG-905 BM-792 1165.7 64.125 33241295.5 1.000 3.3 UM/APG-906 BM-957 1179.7 45.3 3333 659.95 0.300 11.7UM/APG-907 BM-962 1193.7 57.41 3389.5 538.15 0.200 18.5 UM/APG-908BM-1075 1113.7 5.7285 178.95 161.45 0.300 15.3 UM/APG-909 BM-1192 1215.3NA NA NA 0.050 60.0 UM/APG-910 BM-1195 1229.3 148.565 666.55 174.7 0.006128.7 UM/APG-911 BM-1197 1245.8 295 447.95 291.6 0.005 366.7 UM/APG-912BM-1244 1273.8 134.52 450.75 356.75 0.300 66.7 UM/APG-913 BM-1261 1409.9106.89 274.45 239.45 27.000 NA UM/APG-914 BM-1252 1395.9 81.085 135.85123.95 6.000 NA

The data show that binding to any of the Bcl isoforms with high affinitywas not necessarily predictive of an effective senolytic agent. Thecompounds designated BM-1075, BM-1195, BM-1197, BM-1244, BM-1261, andBM-1252 all had binding affinities (Ki) for the Bcl isoforms that werein the nanomole to picomole range. However, in the assay to determineeffective concentration for killing the cells (EC₅₀), some of thesemolecules, such as BM-1244, BM-1261, and BM-125, were 60 to 5,000-foldless potent than the compounds ultimately chosen for development.

The compounds with the best senolytic activity, BM-1195 and BM-1197,were potent in the nanomole range. There was a wide range of specificityfor senescent cells (SI) determined for the various compounds, rangingfrom 1.0 (non-specific) to over 300. The best compound in terms of bothpotency and specificity was BM-1197, with BM-1195, BM-1244, BM-1105, andBM-1075 also being of interest.

In accordance with the data, the following deductions were made withrespect to the chemical structure. This substructure was at the core ofeffective compounds:

with the R₃, R₄, and F groups being optional. The —SO₂CF₃ in BM-1197 isinfluential, but could be substituted with groups having similarproperties, such as —NO₂ in BM-1075. The —SO₂R′ group is influential,although R′ could be varied from —CH₃ to other short-chain alkyl groups.The aryl —S—C₆H₅ group is also influential, although it couldpotentially have neutral substituents. With respect to the followingpart of the structure:

R₁ can be several short-chain alkyl groups, and X₁ can be varied (Cl inBM-1197; F in BM-1195). The following part of BM-1197:

appears to be forgiving in form, with a range of alternativesubstructures being effective for many purposes of this invention.

These and other deductions lead to the drawing of the generic structureshown earlier as Formula VI and Formula VII.

Example 4: Effect of Senolytic Agents on Senescent Retinal VascularCells

The ability of candidate agents to eliminate senescent cells orsenescent-like retinal endothelial cells was measured directly in thefollowing assay.

Human retinal microvascular endothelial cells (HRMEC) were obtained fromNeuromics with the designation HEC09. The cells were initiallymaintained and propagated at <75% confluency in ENDO-Growth Media(Neuromics, Edina Minn.) at 3% O2, 10% CO2, and ˜95% humidity. The cellswere divided into three groups: irradiated cells (cultured for 7 daysafter irradiation prior to use), proliferating normal cells (cultured atlow density for one day prior to use), and quiescent cells (cultured toconfluency over 4 days).

On day 0, the irradiated cells were prepared as follows, HRMEC cellswere covered with 3 mL of TrypLE trypsin-containing reagent(Thermofisher Scientific, Waltham, Mass.) and incubated for 5 min untilthe cells rounded up and began to detach from the plate. Cells weredispersed, counted, and prepared in medium at a concentration of 100,000cells per mL. This cell suspension was placed in T175 flasks at adensity of 100,000 cells per mL and irradiated at 10-15 Gy. Followingirradiation, the cells were plated at 100 μL in 96-well plates. On days1, 3, 6, the medium in each well was aspirated and replaced with freshmedium.

On day 3, the quiescent healthy cells were prepared as follows. Healthy,non-senescent HRMEC cells were released from the culture flask byincubation with 3 mL of TrypLE trypsin-containing reagent (ThermofisherScientific, Waltham, Mass.) and incubated for 5 min until the cellsrounded up and began to detach from the plate. Cells were dispersed,counted, and prepared in medium at a concentration of 80,000 cells permL. 100 μL of the cells was plated in each well of a 96-well plate.Medium was changed on days 1, 3, 6, and 10.

On day 6, the proliferating healthy cell population as prepared asfollows. Healthy non-senescent HRMEC cells were washed, covered with 3mL of TrypLE and cultured for 5 minutes until the cells rounded up andbegan to detach from the plate. Cells were dispersed, counted, andprepared in medium at a concentration of 40,000 cells per mL. 100 μL ofthe cells was plated in each well of a 96-well plate.

On day 7, candidate senolytic agents were combined with the cells asfollows. A DMSO dilution series of each test compound was prepared at200 times the final desired concentration in a 96-well PCR plate.Immediately before use, the DMSO stocks were diluted 1:200 intoprewarmed complete medium. Medium was aspirated from the cells in eachwell, and 100 μL/well of the compound containing medium was added.

The candidate senolytic agents were cultured with the cells for 3 days.The assay system used the properties of a thermostable luciferase toenable reaction conditions that generate a stable luminescent signalwhile simultaneously inhibiting endogenous ATPase released during celllysis. At the end of the culture period, the plates were removed fromthe incubator and allowed to equilibrate at room temperature for 20minutes then 100 μL of CellTiter-Glo® reagent (Promega Corp., Madison,Wis.) was added to each of the wells. The cell plates were placed for 30seconds on an orbital shaker and then allowed to stand at roomtemperature for 10 minutes before measuring luminescence. Theluminescence readings were normalized to determine % cellsurvival/growth and plotted against test compound concentrations, andpotencies (IC₅₀ values) were determined by non-linear curve fitting inGraphpad Prism®.

FIG. 7 shows the results. The concentration-response curve for anexample test compound, demonstrates sensitivity of senescent HRMEC cellsurvival to incubation with a senolytic molecule. These data show thatsenolytic agents are capable of eliminating senescent HRMEC cells inculture.

Example 5: Effect of Senolytic Agents on Senescent Cells from the Eye

The ability of candidate agents to eliminate senescent cells orsenescent-like cells was measured directly in the following assay.

Human retinal pigmented epithelial (RPE) cells were obtained from Lonzawith the designation 194987. The cells were initially maintained andpropagated at <75% confluency in RPE media defined by Sonoda et al. in2009 with 5% FBS and Pen/Strep in an atmosphere of 3% O2, 10% CO2, and˜95% humidity. The cells were divided into three groups: irradiatedcells (cultured for 12 days after irradiation prior to use),proliferating normal cells (cultured at low density for two day prior touse), and quiescent cells (cultured to confluency over 12 days).

On day 0, the irradiated cells were prepared as follows, RPE cells werecovered with 3 mL of TrypLE trypsin-containing reagent (ThermofisherScientific, Waltham, Mass.) and incubated for 5 min until the cellsrounded up and began to detach from the plate. Cells were dispersed,counted, and prepared in medium at a concentration of 100,000 cells permL. This cell suspension was placed in T175 flasks at a density of100,000 cells per mL and irradiated at 10-15 Gy. Following irradiation,the cells were plated at 100 μL in 96-well plates. On days 1, 3, 6, and10 the medium in each well was aspirated and replaced with fresh medium.

On day 0, the quiescent healthy cells were prepared as follows. Healthy,non-senescent RPE cells were released from the culture flask usingtrypsin, covered with 3 mL of TrypLE trypsin-containing reagent(Thermofisher Scientific, Waltham, Mass.) and incubated for 5 min untilthe cells rounded up and began to detach from the plate. Cells weredispersed, counted, and prepared in medium at a concentration of 100,000cells per mL. 100 μL of the cells was plated in each well of a 96-wellplate. Medium was changed on days 1, 3, 6, and 10.

On day 10, the proliferating healthy cell population as prepared asfollows. Healthy non-senescent RPE cells were washed, covered with 3 mLof TrypLE and cultured for 5 minutes until the cells rounded up andbegan to detach from the plate. Cells were dispersed, counted, andprepared in medium at a concentration of 30,000 cells per mL. 100 μL ofthe cells was plated in each well of a 96-well plate.

On day 12, candidate senolytic agents were combined with the cells asfollows. A DMSO dilution series of each test compound was prepared at200 times the final desired concentration in a 96-well PCR plate.Immediately before use, the DMSO stocks were diluted 1:200 intoprewarmed complete medium. Medium was aspirated from the cells in eachwell, and 100 μL/well of the compound containing medium was added.

The candidate senolytic agents were cultured with the cells for 3 days.The assay system used the properties of a thermostable luciferase toenable reaction conditions that generate a stable luminescent signalwhile simultaneously inhibiting endogenous ATPase released during celllysis. At the end of the culture period, the plates were removed fromthe incubator and allowed to equilibrate at room temperature for 20minutes then 100 μL of CellTiter-Glo® reagent (Promega Corp., Madison,Wis.) was added to each of the wells. The cell plates were placed for 30seconds on an orbital shaker and then allowed to stand at roomtemperature for 10 minutes before measuring luminescence. Theluminescence readings were normalized to determine % cellsurvival/growth and plotted against test compound concentrations, andpotencies (IC₅₀ values) were determined by non-linear curve fitting inGraphpad Prism.

FIG. 8 shows the results. The concentration-response curve for anexample test compound, demonstrates sensitivity of senescent RPE cellsurvival to incubation with a senolytic molecule. In contrast, the IC₅₀for proliferating, non-senescent RPE is higher than that determined forthe senescent cells. These data show that senolytic agents are capableof eliminating senescent RPE cells in culture.

Example 6: Efficacy of Compounds in an Animal Model of IschemicRetinopathy

The efficacy of model compound UBX1967 was studied in the mouseoxygen-induced retinopathy (OIR) model, which provides an in vivo modelof retinopathy of prematurity (ROP) and diabetic retinopathy.

C57Bl/6 mouse pups and their CD1 foster mothers were exposed to a highoxygen environment (75% O₂) from postnatal day 7 (P7) to P12. At P12,animals were injected intravitreally with 1 μl test compound (200, 20,or 2 uM) formulated in 1% DMSO, 10% Tween-80, 20% PEG-400, and returnedto room air until P17. Eyes were enucleated at P17 and retinas dissectedfor either vascular staining or qRT-PCR. To determine avascular orneovascular area, retinas were flatmounted, and stained with isolectinB4 (IB4) diluted 1:100 in 1 mM CaCl₂. For quantitative measurement ofsenesecence markers (e.g., Cdkn2a, Cdkn1a, Il6, Vegfa), qPCR wasperformed. RNA was isolated and cDNA was generated byreverse-transcription, which was used for qRT-PCR of the selectedtranscripts.

FIGS. 9A and 9B show that intravitreal (IVT) administration UBX1967resulted in statistically significant improvement in the degree ofneovascularization and vaso-obliteration at all dose levels.

FIGS. 10A and 10B show the relative abundance of several transcriptsassociated with senescence (p16, pai1) and human disease (vegf).Treatment with UBX1967 resulted in a 58%, 35%, and 24% reduction in p16,pai1, and vegf, respectively. Senescence-associated β-galactosidase(SA-βGal) activity was reduced by 17% after administration of UBX1967.

These results show that a single ocular injection of UBX1967 canfunctionally inhibit pathogenic angiogenesis and promote vascular repairin this key OIR disease model. We believe that efficacy of UBX1967 inthe OIR model is due to elimination of senescent cells and accompanyingSASP that propagates senescence in retinal cells and promotesneovascularization of retinal vessels.

Example 7: Efficacy of Compounds in an Animal Model of Diabetes InducedRetinopathy

The efficacy of UBX1967 was studied in a mouse model of diabeticretinopathy, by a single administration of streptozotocin (STZ).

C57BL/6J mice of 6- to 7-week were weighted and their baseline glycemiawas measured (Accu-Chek, Roche). Mice were injected intraperitoneallywith STZ (Sigma-Alderich, St. Lois, Mo.) for 5 consecutive days at 55mg/Kg. Age-matched controls were injected with buffer only. Glycemia wasmeasured again a week after the last STZ injection and mice wereconsidered diabetic if their non-fasted glycemia was higher than 17 mM(300 mg/dL). STZ treated diabetic C57BL/6J mice were intravitreallyinjected with 1 μl of UBX1967 (2 μM or 20 μM, formulated as a suspensionin 0.015% polysorbate-80, 0.2% Sodium Phosphate, 0.75% Sodium Chloride,pH 7.2) at 8 and 9 weeks after STZ administration. Retinal Evans bluepermeation assay was performed at 10 weeks after STZ treatment.

FIGS. 11A and 11B show preliminary results for this protocol. Retinaland choroidal vascular leakage after intravitreal (IVT) administrationUBX1967 improved in vascular permeability at both dose levels.

Example 8: Efficacy of Compounds in an Animal Model of Glaucoma

Administration of bleomycin, a DNA damaging agent, to the anteriorchamber of the mouse or rabbit eye leads to cellular senescence, asdetected by the induction of p16 transcript in the trabecular meshwork.

To induce a senescent phenotype in the trabecular meshwork in vivo,C57Bl/6 mice (aged 8 to 10 weeks) were injected intracamerally with 2 μLof 0.0075 U bleomycin sulfate. In the rabbit, 30 μL of 0.0075 Ubleomycin sulfate were injected intracamerally in New Zealand whiterabbits. Eyes were enucleated 14 days post-bleomycin injury andTM-enriched samples were micro-dissected. To determine change insenescent cells, RNA was isolated from TM and qPCR analysis was done toassess the effect of bleomycin on p16 mRNA levels.

FIGS. 12A and 12B show elevated relative expression of p16 at 14 daysafter intracameral (IC) injection of bleomycin in the right (OD) eyerelative to the PBS-injected left (OS) eye of the test animals. Thismodel can also be used to assess whether a test compound ispharmacologically capable of reducing or ameliorating the increasedintraocular pressure that is a hallmark of primary open angle glaucoma(POAG).

Example 9: Senescent Cells in the Trabecular Meshwork of Tissue Samplesfrom Humans Having POAG

To obtain evidence for the presence of senescent cells in human patientshaving primary open angle glaucoma (POAG), tissue samples obtained fromaffected patents were stained for p16, a marker of senescent cells.

Donor globes were procured prospectively from eye banks and placedimmediately into Davidson's reagent to fix for at least 48 hours. Afterfixation, the solution was exchanged for 70% ethanol. Tissue was thenplaced in histology cassettes and processed for paraffin infiltrationand embedding. Paraffin blocks were mounted on a microtome and sectionedto 5-9 μm thickness. Cut tissue sections were placed into a 45° C. waterbath, and Superfrost Plus™ microscope slides were used to pick upsections, wicking away excess water with a Kimwipe™ tissue. Slides werethen placed upright to dry for 30 min at room temperature, followed byincubation overnight in a 37° C. incubator. Slides are baked for 15-20min at 60° C. and then allowed to cool to room temperature.

Slides were dewaxed by incubation in BOND Dewaxing Solution at 70° C.for 30 s. Slides were then rehydrated by serial incubations indecreasing concentrations of ethanol as follows: twice in 100% ethanolfor 5 min each, twice in 90% ethanol for 2 min each, twice in 75%ethanol for 2 min each, twice in 50% ethanol for 2 min each, and thenrinsed under water for 4 min. Slides were then washed twice inTris-buffered saline supplemented with 0.1% Triton-X 100 (TBST).

Antigen retrieval was performed by incubation in BOND ER1 solution(sodium citrate buffer, pH 6.0) at 95° C. for 20 min. Slides wereincubated in 3% H₂O₂ for 10 min at room temperature, followed by washingtwice in TBST.

Slides were incubated for 30 min at room temperature with p16 primaryantibody (Roche, #9517), diluted 1:2 in TBST. After primary antibodyincubation, slides were washed twice with TBST prior to incubation withthe rabbit-anti-mouse secondary antibody for 20 min at room temperature.Slides were washed twice with TBST and incubated for 20 min at roomtemperature in HRP-conjugated secondary antibody. After antibodyincubation, slides were washed twice with TBST and AEC chromogen wasused for detection, prepared per manufacturer protocol, and color wasallowed to develop for approximately 20 min. Slides were then rinsedthree times in water, before incubation in Harris' hematoxylin for 20 s.Slides were washed thoroughly in water, and coverslips were applied tothe slides over Vectashield™ mounting media (Vector Labs, BurlingameCalif.) and allowed to dry before imaging.

FIGS. 13A and 13B are representative images of p16 immunohistochemistryon human eye tissue obtained from donors diagnosed with POAG. Cellspositive for expression of p16 appear with dark staining in the cells inand around the trabecular meshwork (circled area).

Example 10: Senescent Cells in Tissue from Humans with Age-RelatedMacular Degeneration (AMD)

To obtain evidence for the presence of senescent cells in AMD, tissuesamples from affected patents were stained for p16.

Donor globes were procured prospectively from eye banks and placedimmediately into Davidson's reagent to fix for at least 48 hours. Afterfixation, the solution was exchanged for 70% ethanol. Tissue was thenplaced in histology cassettes and processed for paraffin infiltrationand embedding. Paraffin blocks were mounted on a microtome and sectionedto 5-9 μm thickness. Cut tissue sections were placed into a 45° C. waterbath, and Superfrost Plus microscope slides were used to pick upsections, wicking away excess water with a Kimwipe. Slides were thenplaced upright to dry for 30 min at room temperature, followed byincubation overnight in a 37° C. incubator. Slides were baked for 15-20min at 60° C. and then allowed to cool to room temperature. Slides weredewaxed using xylene or xylene substitute (e.g., Histoclear™) for 4 min,repeated for a total of 3 incubations. Slides were then rehydrated byserial incubations in decreasing concentrations of ethanol as follows:twice in 100% ethanol for 5 min each, twice in 90% ethanol for 2 mineach, twice in 75% ethanol for 2 min each, twice in 50% ethanol for 2min each, and then rinsed under water for 4 min.

Antigen retrieval was performed by incubation in acidic sodium citratebuffer at 120° C. for 3 min (or 95° C. for 20 min) using a steamer orpressure cooker. Slides were cooled to room temperature for 15 minfollowing by washing twice in Tris-buffered saline supplemented with0.1% Triton-X 100 (TBST) for 2 min each. Slides were washed twice againwith TBST for 5 min each prior to blocking for 1 h at room temperaturein TBST containing 5% normal serum, from the species of origin of thesecondary antibody to be used. After blocking, the slides were drainedand excess solution was wiped away. Slides were incubated overnight at4° C. (or for 2 h at room temperature) with p16 primary antibody diluted1:2 in TBST. After primary antibody incubation, slides were washed twicewith TBST for 5 min each, then treated with 3% H₂O₂ in TBST for 15 min.Slides were then washed twice with TBST for 5 min each prior tosecondary antibody incubation. Slides were incubated for 1 h at roomtemperature in HRP-conjugated secondary antibody. After antibodyincubation, slides were washed three times with TBS for 5 min each. AECchromogen was used for detection, prepared per manufacturer protocol,and color was allowed to develop for approximately 20 min. Slides werethen rinsed in water for 5 min, before counterstaining with hematoxylin.Coverslips were applied to the slides over Vectashield mounting media(Vector Labs, Burlingame Calif.) and allowed to dry before imaging.

FIGS. 14A and 14B are representative images of p16 immunohistochemistryon human eye tissue obtained from donors diagnosed with AMD. Cellspositive for expression of p16 appear with dark staining in areas withobvious disease pathology (circled area).

Example 11: Clinical Assessment

This example provides an outline for a pre-clinical or clinical trial toevaluate the safety and efficacy of a senolytic agent for the treatmentof ophthalmic conditions.

The senolytic is administered to subjects in the trial by standardintravitreal (ITV) administration technique, with the eye washed anddraped in usual sterile fashion following pre-injection IOP measurement.Topical anesthesia is applied and a lid speculum placed for adequateexposure. The injection quadrant is chosen by the treating physician,and a location for the injection measured at 3 to 4 mm posterior to thecorneo-scleral limbus. A 28-32-gauge needle is used to administer a 0.05mL to 0.1 mL injection of the compound. The lid speculum is removed atthe conclusion of the injection procedure. Depending on the nature ofthe condition, potentially suitable intra- or peri-ocular deliverymethods include intravitreal, intracameral, posterior juxtascleral,subconjunctival or suprachoroidal injection.

Following the treatment, subjects are evaluated to determine whethersymptoms or signs of the ophthalmic condition are improved, relative tosubjects in a control group, using commonly available tests of ocularstructure and function (supra).

Example 12: Combination Therapy

This example illustrates the clinical use of a senolytic agent incombination with standard-of-care anti-VEGF therapy for VEGF-relatedocular disease.

Subjects are recruited into the study that have wet AMD, and are alreadyundergoing anti-VEGF therapy, or that have a need to initiate VEGFtherapy. First administration of a senolytic compound is done on thesame day as one of the regular administrations of anti-VEGF therapies.There are no expected contraindications to the use of ophthalmictherapies in conjunction with the senolytic compound. The senolyticagent is administered first by standard intravitreal (ITV)administration. Intraocular pressure (IOP) is measured at 15 and 30minutes post-injection. When IOP is normalized to pre-injection levels,the anti-VEGF therapy (for example aflibercept 2.0 mg in 0.05 mL) isadministered second. The eye is washed and draped in usual sterilefashion, as an entirely new sterile procedure from the first injection.Topical anesthesia is given and a lid speculum placed for adequateexposure. The injection quadrant is chosen by the treating physician,and a location for the injection measured at 3 to 4 mm posterior to thecorneo-scleral limbus. A 28-32-gauge needle is used to administer a 0.05mL injection of the compound. The lid speculum is removed at theconclusion of the injection. IOP is monitored at 5, 15 and 30 minutesfollowing the second ITV injection.

Safety and efficacy of the combined treatment is assessed as follows. Inthe immediate post-injection period, the eye is monitored for any acuteloss of vision or increase in intraocular pressure. At 30 minutespost-injection if vision and IOP are stable, the patient is dischargedfrom the clinic under the standard post-procedure instructions of thetreating physician. The patient is instructed to call immediately forthe development of any change in vision or onset of pain or redness inthe injected eye.

In the setting of this disease example of wet AMD, follow up evaluationis scheduled within one week to one month of treatment. Follow-upevaluation includes visual acuity and IOP measurement, monitoring forany improvement or decline since the therapy was administered. Theanterior and posterior segment of the eye is examined and standardancillary testing is performed, including but not limited tophotography, IV fluorescein angiography and Optical Coherence Tomography(OCT).

Response to the senolytic is documented using this combination ofclinical examination, functional and structural testing described above.Further follow-up is scheduled based on patient response. If thesenolytic is given in conjunction with an anti-VEGF agent, the physiciancontinues to follow the visit and treatment administration scheduleassociated with that agent.

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The several hypotheses presented in this disclosure provide a premise byway of which the reader may understand the invention. This premise isprovided for the enrichment and appreciation of the reader. Practice ofthe invention does not require detailed understanding or application ofthe hypothesis. Except where stated otherwise, features of thehypothesis presented in this disclosure do not limit application orpractice of the claimed invention. For example, except where theelimination of senescent cells is explicitly required, the compounds ofthis invention may be used for treating the conditions describedregardless of their effect on senescent cells. Although many of theophthalmic conditions referred to in this disclosure occur predominantlyin older patients, the invention may be practiced on patients of any agehaving the condition indicated, unless otherwise explicitly indicated orotherwise required.

While the invention has been described with reference to the specificexamples and illustrations, changes can be made and equivalents can besubstituted to adapt to a particular context or intended use as a matterof routine development and optimization and within the purview of one ofordinary skill in the art, thereby achieving benefits of the inventionwithout departing from the scope of what is claimed.

The invention claimed is:
 1. A method of inhibiting vaso-obliterationthat leads to vision loss in an eye of a subject in need thereof,comprising administering to the eye a pharmaceutical compositioncomprising an effective amount of a compound, wherein the compound isBM-1197.
 2. The method of claim 1, wherein the amount of the compound inthe pharmaceutical composition that is administered to the eye is alsoeffective in inhibiting pathogenic angiogenesis in the eye.
 3. Themethod of claim 1, wherein the amount of the compound in thepharmaceutical composition that is administered to the eye is alsoeffective in inhibiting retinal neovascularization in the eye.
 4. Themethod of claim 1, wherein the amount of the compound in thepharmaceutical composition that is administered to the eye is alsoeffective in inhibiting retinal or choroidal vascular leakage in theeye.
 5. The method of claim 1, wherein the amount of the compound in thepharmaceutical composition that is administered to the eye is alsoeffective in promoting repair of functional vasculature in the eye.